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Molecular Endocrinology 21 (4): 963-971
Copyright © 2007 by The Endocrine Society

Maximal Activity of the Luteinizing Hormoneß-Subunit Gene Requires ß-Catenin

Travis B. Salisbury, April K. Binder, Jean C. Grammer and John H. Nilson

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164

Address all correspondence and requests for reprints to: John H. Nilson, School of Molecular Biosciences, Fulmer 639A, Washington State University, Pullman, Washington 99164-4660. E-mail: jhn{at}wsu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH regulates expression of LHB via transcriptional regulation of early growth response 1 (EGR1), an immediate early gene that encodes a zinc-finger DNA-binding protein. EGR1 interacts functionally with the orphan nuclear receptor steroidogenic factor 1 (SF1) and pituitary homeobox 1, a member of the paired-like homeodomain family. The functional synergism of this tripartite interaction defines the maximal level of LHB transcription that can occur in response to GnRH. Results presented herein provide new evidence that the interaction between SF1 and EGR1 also requires ß-catenin, a transcriptional coactivator and member of the canonical Wnt signaling pathway. For instance, targeted reduction of ß-catenin attenuates activity of a GnRH-primed LHB promoter. Additional gene reporter assays indicate that overexpression of ß-catenin, or its targeted reduction by small interfering RNA, modulates activity of both SF1 and EGR1 as well as their functional interaction. ß-Catenin coimmunoprecipitates with SF1. Moreover, an SF1 mutant that lacks a ß-catenin binding domain has compromised transcriptional activity and fails to interact synergistically with EGR1. Finally, GnRH promotes ß-catenin colocalization with SF1 and EGR1 on the endogenous mouse Lhb promoter-regulatory region. Taken together, these data suggest that ß-catenin binds to SF1 and that this interaction is required for subsequent functional interaction with EGR1. Thus, these data identify ß-catenin as a new and required member of the basal transcriptional complex that allows the LHB promoter to achieve maximal activity in response to GnRH.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
REPRODUCTIVE FUNCTION and fertility require appropriate synthesis and release of LH (1, 2, 3). Appropriate level of LH is achieved through highly interactive feedback loops that regulate secretion of the hormone as well as transcription of the two genes that encode its subunits: common glycoprotein {alpha} (CGA/Cga) and LH ß (LHB/Lhb). Secretion of LH occurs in pulses that are driven by hypothalamic secretion of GnRH and counterbalanced by gonadal steroids (4). GnRH and steroids also regulate transcription of both genes (5, 6, 7, 8). Herein, we focus on a complex set of interactions that are required for maximal transcription of LHB/Lhb in the presence of GnRH.

Regulated LHB/Lhb gene expression requires combinations of regulatory elements that cluster in the proximal and distal regions of the promoter (8). The proximal domain is conserved across all mammals and contains a single central element that binds pituitary homeobox 1 (PITX1) (8, 9, 10). The single PITX1 element is flanked by pairs of elements that bind steroidogenic factor 1 (SF1) and early growth response 1 (EGR1) (11, 12, 13, 14, 15, 16). Gonadotrope-specific expression of LHB/Lhb genes also occurs through contributions provided by a distal regulatory domain. This domain displays species-specific variation and includes arrays of elements ranging from tandem binding sites for nuclear factor Y (17) to a tripartite composite element that contains two specificity protein 1-binding sites flanking a single CArG response element (18, 19).

GnRH stimulates LHB/Lhb gene expression via transcriptional regulation of EGR1 (11, 20, 21, 22, 23). The transcriptional contribution provided by GnRH induction of EGR1 is further amplified by subsequent functional interactions with SF1 and PITX1 (10, 11, 12, 13, 16, 21, 24). Indeed, physical interaction between these three transcription factors underlies their synergistic action (10, 11).

In contrast to EGR1, levels of SF1 and PITX1 remain unaffected by GnRH (11, 21). Consequently, contributions from SF1 and PITX1 set the basal transcriptional tone of the LHB promoter as well as serve as key amplifiers of GnRH signaling through their functional synergism with EGR1 (8, 11, 13, 21, 24).

Whereas it is clear that EGR1 is a primary downstream target of GnRH (20), it alone is insufficient to allow for adequate transcription of the LHB/Lhb gene. This is best illustrated by recent studies with mice that harbor gonadotrope-specific, Cre recombinase-mediated deletion of SF1 (25, 26). Mice lacking SF1 in gonadotropes of the pituitary were hypogonadal with undetectable levels of Lhb and Fshb mRNA when assayed by PCR (25, 26). Hence, SF1 plays an essential role in maximizing the transcriptional signal provided by GnRH-induced EGR1.

In this study, we asked whether the functional interplay between SF1 and EGR1 requires a coactivator. The impetus for this line of investigation stems from two reports showing that ß-catenin acts as a coactivator of SF1 when it transduces Wnt signals to Dax1 (officially NR0B1) and Inhibin {alpha} (officially Inha) promoters (27, 28). Coactivation occurs through the binding of ß-catenin to a cluster of amino acids (235–238) located in the first helix of the putative ligand-binding domain of SF1 that also contains the activation function 1 domain (28). Herein we report a new role for ß-catenin in regulation of LHB/Lhb gene expression in gonadotropes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Maximal Transcriptional Response of the LHB Promoter to GnRH Requires ß-Catenin
GnRH induction of EGR1 maximizes expression of the endogenous Lhb gene and LHB/Lhb reporter constructs in the LßT2 gonadotrope cell line (11, 13, 21). Accordingly, to assess the requirement for ß-catenin, we targeted its reduction in LßT2 cells treated with either vehicle or GnRH via two different strategies and then assayed activity of a cotransfected LHB reporter construct.

The first strategy involved overexpression of AXIN. This protein promotes degradation of cellular ß-catenin levels (29, 30, 31). Consequently, we cotransfected LßT2 cells with LHB (LHB-luciferase) and phRG-B-Renilla reporter constructs along with a cytomegalovirus (CMV)-Axin expression vector. Controls included cells treated with vehicle instead of GnRH and cells cotransfected with an empty expression vector. Post transfection, LßT2 cells were treated for 24 h with either vehicle or 10 nM GnRH.

As expected, activity of the LHB reporter is marginal in the absence of GnRH (Fig. 1AGo, lane 1) making detection of an AXIN effect on basal activity of the reporter problematic (Fig. 1AGo, lane 2). In contrast, AXIN strongly suppressed LHB promoter activity in LßT2 cells treated with GnRH (P < 0.01, Fig. 1AGo, compare lanes 3 and 4). Although it is clear that overexpression of AXIN prevents the LHB promoter from achieving maximal activity after treatment with GnRH, we were unable to confirm reduction of ß-catenin in these transient expression assays. Nevertheless, this outcome is consistent with the possibility that changes in ß-catenin levels modulate activity of the LHB promoter.


Figure 1
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  		Fig. 1. Maximal Transcriptional Response of the LHB Promoter to GnRH Requires ß-Catenin

A, LßT2 cells were transiently co-transfected with –779/+10 bovine LHB promoter-luciferase (200 ng) and phRG-B-Renilla reporter constructs (10 ng) and an expression vector encoding AXIN (100 ng) and then treated with either GnRH (10 nM) or vehicle. NFY, Nuclear factor Y. Luciferase activities for each data point were normalized against Renilla activities. To calculate fold change within an experiment, LHB promoter activities were expressed relative to the maximal amount of LHB promoter activity, assigned a value of 1.0 in cells transfected with an empty CMV expression vector, and treated with GnRH. Values were then averaged from three experiments performed in triplicate. Data shown are the means ± SEM of these three experiments. *, P < 0.01. B, LßT2 cells were transiently transfected using a reverse transfection protocol (32 ) with –779/+10 bovine LHB promoter-luciferase vector and 100 nM of nontargeting control interfering RNA (siRNAc) or ß-catenin interfering RNA (siRNAß) and subsequently treated with either vehicle or GnRH (10 nM). Luciferase activities for each data point were normalized to total amount of protein. Fold change represents LHB promoter activities expressed relative to the maximal amount of LHB promoter activity, assigned a value of 1.0 in cells transfected with siRNAc, and treated with GnRH. Values were averaged from three experiments performed at least in duplicate. Data shown are the means ± SEM of three experiments. *, P < 0.01. C, Transfection lysates from experiments in Fig. 2BGo were probed by immunoblot analysis of ß-catenin and AKT, the latter as a loading control. Results shown are representative of two experiments. ß-cat, ß-Catenin.

 
To more directly examine a role for ß-catenin in GnRH regulation of LHB promoter activity, we used a pool of four different small interfering RNAs (siRNAs) specific for ß-catenin (SMARTpool; Dharmacon, Lafayette, CO) to reduce its levels in LßT2 cells. This experiment involved a different transfection paradigm than that used in the AXIN overexpression studies. Pools of siRNA were introduced by reverse transfection (32) along with the LHB-luciferase construct over a 72-h period. Cells were then treated with 10 nM GnRH for 24 h and assayed for luciferase activity. To control for specificity, LßT2 cells were also reverse transfected with a pool of nonspecific siRNA (Dharmacon).

In this reverse transfection paradigm, the siRNA specific for ß-catenin (siRNAß) reduced basal activity of the LHB reporter by approximately 50% when compared with cells treated with the control siRNA (Fig. 1BGo; compare lanes 1 and 2). This reduction in the absence of GnRH suggests that ß-catenin contributes to basal promoter activity presumably through an interaction with one or several of the DNA-binding proteins that populate this region.

The pool of siRNA specific for ß-catenin siRNAß also significantly attenuated the activity of LHB reporter in the presence of GnRH (P < 0.01; Fig. 1BGo; compare lanes 3 and 4). The extent of the reduction in the presence of GnRH was 67%, slightly greater than that observed in the absence of GnRH. Whereas it is tempting to suggest that ß-catenin also alters GnRH responsiveness, it is difficult to make this conclusion based on a single time point. It is clear, however, that treatment with the ß-catenin-specific siRNA prevents the LHB reporter from achieving maximal activity when exposed to GnRH.

Levels of total cellular ß-catenin were also examined by immunoblot (Fig. 1CGo). Specific siRNA-mediated reduction of ß-catenin was not detected in LßT2 cells treated with vehicle (Fig. 1CGo; lane 2 vs. lane 1). In contrast, specific siRNA reduction of ß-catenin was readily detected in LßT2 cells treated with GnRH. The reason for this apparent difference in siRNA efficiency remains unclear but could reflect specific effects of GnRH that facilitate one or several members of the siRNA pathway. Nevertheless, the siRNA-mediated reduction of ß-catenin observed in the presence of GnRH, coupled with the loss of LHB-reporter activity, complements the results obtained with the AXIN paradigm. Together, these results suggest that ß-catenin may be an essential determinant for establishing the basal transcriptional tone of the LHB promoter.

ß-Catenin Enhances Activity of SF1 and EGR1
ß-Catenin functions as a coactivator for DNA-bound transcription factors in the canonical WNT signaling pathway (33). Therefore, it seemed likely that ß-catenin could contribute to LHB promoter activity by interacting with any one of the primary transcriptional components of the proximal regulatory region (8). In LßT2 cells, overexpression of either EGR1 or SF1 increases activity of a LHB reporter (see below). In contrast, overexpression of PITX1 has no effect (data not shown). Consequently, we focused on examining whether ß-catenin modulates activity of either SF1 or EGR1.

Transactivation properties of ß-catenin, SF1, and EGR1 were assessed by transient cotransfection assays in LßT2 cells maintained in the absence of GnRH (Fig. 2Go). In this state, levels of EGR1 are extremely low, and the endogenous Lhb gene is nearly silent (11). Because EGR1 is induced by GnRH, overexpression of EGR1 acts as surrogate of the neurohormone. In contrast to EGR1, levels and apparent activity of SF1 are unaffected by GnRH (11). Thus, effects of transfected SF1 depend on whether the levels of endogenous orphan receptor are limiting with respect to activity of the LHB promoter. Because LßT2 cells contain ample ß-catenin, detecting changes conferred by transfected ß-catenin also depends on whether endogenous levels of the protein are limiting or in excess with respect to the cotransfected LHB promoter.


Figure 2
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  		Fig. 2. ß-Catenin Enhances Activity of EGR1 and SF1

Transient cotransfection assays were performed in LßT2 cells with –779/+10 bovine LHB promoter-luciferase and phRG-B-Renilla reporter constructs. NFY, Nuclear factor Y. The activity of these reporter constructs was assayed upon their cotransfection with expression vectors encoding either {Delta} 90 ß-catenin (100 ng), SF1 (20 ng), or EGR1 (10 ng). Luciferase activities for each data point were normalized against Renilla activities. To calculate fold change, LHB promoter activities were expressed relative to the amount of LHB promoter activity in the control group (cells transfected with empty CMV expression vectors and reporter vectors). Data shown are the means ± SEM from three experiments performed in triplicate. Significant differences (P < 0.01) are indicated between groups that do not share any lowercase letters. ß-cat, ß-Catenin.

 
Overexpression of a constitutively active form of ß-catenin [{Delta}90; (34)] only marginally increased activity of the LHB reporter (Fig. 2Go, lanes 1 and 2). This result would be expected if endogenous levels of ß-catenin are close to that required for a maximal transcriptional contribution. In contrast, overexpression of either SF1 or EGR1 increased activity of the LHB promoter (P < 0.01; Fig. 2Go, lanes 3 and 5) indicating that their endogenous levels are limiting. Interestingly, {Delta} 90 ß-catenin significantly enhanced the transcriptional effects of both SF1 (P < 0.01; Fig. 2Go, lanes 3 and 4) and EGR1 (P < 0.01; Fig. 2Go, lanes 5 and 6). This result suggests that endogenous levels of ß-catenin became limiting with the overexpression of either SF1 or EGR1. Together, these data underscore the notion that ß-catenin acts as coactivator for either one or both of these DNA-binding proteins.

The Functional Interaction between EGR1 and SF1 Also Requires ß-Catenin
Our finding that ß-catenin enhances the transcriptional effects of either EGR1 and SF1 suggests that the coactivator may also be required for the functional interaction that occurs when both factors are bound to the proximal LHB promoter. Thus, we explored the importance of ß-catenin in modulating the interplay between SF1 and EGR1 by employing an siRNA strategy similar to that described earlier (Fig. 1Go).

LßT2 cells maintained in the absence of GnRH were cotransfected with either pooled control or ß-catenin-specific siRNA and with expression vectors encoding either SF1 or EGR1 or with both expression vectors. Transactivation of the LHB reporter by SF1 was significantly reduced when cells were transfected with siRNA specific for ß-catenin (P < 0.01; Fig. 3Go, lanes 3 and 4). EGR1 activity was also reduced in the presence of siRNA specific for ß-catenin relative to cells cotransfected with EGR1 and control siRNA (P < 0.01; Fig. 3Go, lanes 5 and 6). Treatment with the ß-catenin-specific siRNA also significantly attenuated the maximal response normally observed upon cotransfection of vectors encoding both SF1 and EGR1 (P < 0.01; Fig. 3Go, lanes 7 and 8). Together, these data suggest that maximal transcriptional activity conferred by the interaction between EGR1 and SF1 requires endogenous ß-catenin.


Figure 3
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  		Fig. 3. siRNA against ß-Catenin Reduces the Activity of EGR1 and SF1

LßT2 cells were transiently cotransfected with –779/+10 bovine LHB promoter-luciferase and either 20 nM of siRNAc or 20 nM of siRNAß with expression vectors encoding either SF1 or EGR1. NFY, Nuclear factor Y. Fold change was calculated analogous to that described in Fig. 2Go. Data shown are the means ± SEM of four experiments performed in triplicate. *, P < 0.01.

 
ß-Catenin Acts through SF1
Whereas the transfection studies described in Figs. 1–3GoGoGo indicate that maximal activity of the LHB promoter requires ß-catenin, they also suggest that both SF1 and EGR1 are potential functional targets of the coactivator. There are, however, steadily emerging reports indicating that SF1 and other steroid receptors bind ß-catenin (28, 35, 36). In contrast, there are no reports to date indicating that ß-catenin binds to EGR1. This led to the consideration that ß-catenin contributions to EGR1 activity may be secondary to a primary interaction that occurs between ß-catenin and SF1. We explored this possibility by determining whether ß-catenin could physically interact with SF1 and whether removal of the known ß-catenin-binding site in SF1 would abrogate activity of the orphan receptor.

Physical interaction between SF1 and ß-catenin was explored by coimmunoprecipitation assays. Nuclear extracts were prepared from LßT2 cells treated with either vehicle or GnRH for 60 min and then subjected to immunoprecipitation with an antibody specific to ß-catenin or control IgG. Immunoprecipitates were then subjected to immunoblot analysis employing antibodies specific for either ß-catenin or SF1. In the absence of GnRH, only a marginal association between ß-catenin and SF1 was observed (Fig. 4Go, lane 3). In contrast, GnRH treatment revealed a clear interaction between SF1 and ß-catenin (Fig. 4Go, lane 6). Concomitant immunoblot analysis of whole-cell lysates also indicated that GnRH treatment increased the accumulation of ß-catenin (~2-fold; n = 3, P < 0.05; data not shown).


Figure 4
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  		Fig. 4. SF1 Physically Associates with ß-Catenin

Nuclear extracts isolated from LßT2 cells treated with either vehicle (lanes 1–3) or 10 nM GnRH (lanes 4–6) for 60 min were immunoprecipitated with either anti-ß-catenin antibodies or control rabbit serum. Precipitates were then subjected to immunoblot blot (IB) analysis with SF1 and ß-catenin antibodies. Results are representative of three separate experiments. ß-cat, ß-Catenin; IP, immunoprecipitation.

 
Coimmunoprecipitation was also performed with an antibody specific for EGR1. Whereas EGR1 was also found in a complex containing ß-catenin when cells were treated with GnRH (data not shown), this would be expected given the known physical interaction between SF1 and EGR1 (11). Consequently, these coimmunprecipitation studies indicate the likelihood that SF1, EGR1, and ß-catenin are found together in the same complex.

Nuclear receptors, including SF1, have been reported to interact with ß-catenin (27, 28, 35, 36). Indeed, Mizusaki and colleagues (28) demonstrated that site-specific replacement with four alanines at amino acids 235–238 in SF1 (SF1 235–4A) prevents its physical interaction with ß-catenin when tested by GST-pull-down assays. This mutation also attenuates the transactivation function of the orphan receptor (28). As a consequence, we examined the impact of this mutant on activity of the LHB reporter in cotransfection assays that also employed vectors encoding SF1 and EGR1.

As observed earlier (Figs. 2Go and 3Go), overexpression of SF1 stimulated LHB promoter activity relative to control transfected cells (P < 0.05; Fig. 5Go, lanes 1 and 2). Transfected EGR1 also enhanced the activity of the LHB reporter vector (P < 0.01; Fig. 5Go, lanes 1 and 4). Coexpression of EGR1 and SF1 resulted in maximal stimulation of LHB promoter activity (P < 0.01; Fig. 5Go, lane 5). In contrast, SF1 235–4A virtually eliminated the functionally synergistic interaction between SF1 and EGR1 (P < 0.01; Fig. 5Go, lanes 5 and 6). In essence, the SF1 mutant acts in a dominant-negative fashion. This result is consistent with the known ability of SF1 235–4A to retain DNA-binding activity and limited functional activity when transfected at high concentrations (28). Thus, even if EGR1 interacts directly with ß-catenin, this interaction cannot compensate for the absence of a ß-catenin domain in SF1. Consequently, the dominant-negative effect of SF1 235–4A reinforces the notion that the synergistic interaction between SF1 and EGR1 requires a specific interaction between ß-catenin and the orphan nuclear receptor.


Figure 5
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  		Fig. 5. The ß-Catenin Binding Site in SF1 Is Required for Functional Synergism between SF1 and EGR1

Transient cotransfection assays were performed in LßT2 cells with –779/+10 bovine LHB promoter-luciferase and phRG-B-Renilla reporter constructs. NFY, Nuclear factor Y. The activity of these reporter constructs was assayed upon their cotransfection with expression vectors encoding either SF1 (20 ng), SF1 235–4A (50 ng), or EGR1 (10 ng). Luciferase activities for each data point were normalized against Renilla activities. To calculate fold change, LHB promoter activity for each experimental group is expressed relative to that of "Control" cells that are arbitrarily set at 1. Data shown are the means ± SEM of three experiments. Significant differences (P < 0.05) are indicated between groups that do not share any lowercase letters.

 
GnRH Enhances the Association of ß-Catenin with the Proximal Region of the Endogenous Lhb Promoter
If ß-catenin is required for maximal expression of the endogenous Lhb gene in LßT2 cells, then it should colocalize to the proximal promoter region. We examined this possibility by performing chromatin immunoprecipitation (ChIP) assays. Immunoprecipitated chromatin was subjected to PCR using two primer sets. One primer set (Fig. 6Go) flanks a genomic fragment of the Lhb promoter containing response elements for EGR1, SF1, and PITX1. The other primer set flanks a 5'-distal region of the mouse Lhb promoter and serves as a control.


Figure 6
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  		Fig. 6. GnRH Enhances the Association of ß-Catenin with the Proximal Region of the Endogenous Lhb Promoter

ChIP assays were performed with chromatin prepared from LßT2 cells before and after a GnRH stimulus (15 and 60 min) and immunoprecipitated with anti-ß-catenin antibodies (lanes 1–6), anti-EGR1 antibodies (lanes 7–12), anti-SF1 antibodies (lanes 13–18), or control rabbit serum (lanes 1–18). Precipitated chromatin was amplified with primers that span response elements for EGR1, SF1, and PITX1 (proximal primer set) and with primers that span a distal fragment of Lhb promoters that lack sites for these transcription factors (distal primer set). To calculate fold change in signals from cells treated with vehicle vs. GnRH for 60 min, arbitrary densitometric units for cells treated with GnRH were expressed relative to values in vehicle-treated cells, arbitrarily assigned a value of 1. Statistical analyses were performed on three distinct experiments performed on different days, and results are presented in text as means ± SEM.

 
Modest amounts ß-catenin (Fig. 6Go, lane 4) and EGR1 (Fig. 6Go, lane 10) were associated with chromatin amplified by the primer set specific for the proximal promoter in the absence of a GnRH stimulus. By 60 min, GnRH increased the quantity of ß-catenin (P < 0.05; 2.74-fold ± 0.6 SEM; Fig. 6Go, lane 6) and SF1 (P < 0.05; 2.8-fold ± 0.48 SEM; Fig. 6Go, lane 18) associated with the proximal region of the Lhb promoter. Consistent with prior reports, GnRH also increased the quantity of EGR1 (P < 0.05; 3.86-fold ± 0.7 SEM; Fig. 6Go, lane 12) associated with the proximal region of the Lhb promoter (24). The binding of these three proteins to this region of the promoter was specific because they were not present on chromatin located approximately 3kb upstream (distal primers) from the transcriptional start site of the Lhb gene (Fig. 6Go, lanes 1–3, 7–9, and 13–15), In addition, normal rabbit IgG failed to precipitate chromatin (Fig. 6Go, lanes 4–6, 10–12, and 16–18). Collectively, these data indicate that ß-catenin colocalizes with EGR1 and SF1 on the endogenous Lhb promoter and that GnRH enhances their colocalization.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Pulses of GnRH dynamically regulate transcription of mammalian LHB/Lhb genes (37, 38, 39). This dynamic regulation, however, represents a secondary response to GnRH mediated by the more rapid induction of EGR1 transcription and protein synthesis (11, 20, 21, 22, 23). Indeed, GnRH controls activity of the EGR1 promoter-regulatory region through a protein kinase C/ERK pathway (11, 20, 22). Because levels of SF1 and PITX1 remain unresponsive to changes in GnRH (11, 21), EGR1 is viewed as the primary determinant of hormone-induced LHB/Lhb transcriptional fluxes. Nevertheless, as noted earlier, GnRH induction of EGR1 is not sufficient for ensuring that LH reaches the necessary levels required for physiological activity in transgenic mice with gonadotropes deficient in SF1 (25, 26). Accordingly, SF1 has emerged as a pivotal synergistic partner of EGR1 that amplifies the response of LHB/Lhb genes to GnRH (13, 16, 21). We have now presented several lines of evidence indicating that activity of SF1 and its subsequent synergistic interaction with EGR1 requires ß-catenin. This establishes a new and essential role for ß-catenin; it is a critical coactivator of SF1 that maximizes transcription of LHB/Lhb genes in response to GnRH-induced changes in EGR1.

We identified ß-catenin as a coactivator of SF1 based on the following observations. Reduction of ß-catenin in LßT2 cells through overexpression of AXIN or through use of a pool of siRNA specific to ß-catenin reduced transcription of an LHB reporter construct to GnRH (Fig. 1Go). Although overexpression of ß-catenin increased the transactivation activity of SF1 and EGR1, as well as their functional interaction (Figs. 2Go and 3Go), we suspect that the response of EGR1 was secondary to the effect of ß-catenin on endogenous SF1. In a reciprocal fashion, siRNA specific for ß-catenin attenuated activity of SF1 and EGR1 as well as their functional interaction (Fig. 3Go). GnRH increases accumulation of ß-catenin (data not shown), and immunoprecipitation studies indicated that endogenous SF1, EGR1, and ß-catenin physically associate in LßT2 cells (Fig. 4Go and data not shown). Because the ß-catenin binding pocket of SF1 is required for transactivation activity of the orphan receptor (Fig. 5Go), we suspect that the association of EGR1 is secondary to a primary interaction that occurs between SF1 and ß-catenin. Although we cannot rule out the possibility that ß-catenin also interacts with EGR1, this seems unlikely given the observation that the SF1 mutant lacking a ß-catenin binding site acts in a dominant-negative fashion and almost completely abolished the functional synergism normally exhibited between SF1 and EGR1 (Fig. 5Go). Finally, GnRH enhanced colocalization of ß-catenin to the endogenous promoter region of the mouse Lhb gene that also binds SF1 and EGR1 (Fig. 6Go). Together, these results support the notion that ß-catenin is required for activity of SF1 and its subsequent functional synergism with EGR1.

The supposition that SF1 requires coactivation to contribute to GnRH regulation of the Lhb promoter was first proposed by Kaiser et al. (13) to explain the concerted interaction between specificity protein 1, SF1, and EGR1. Although unidentified, they proposed that a putative cofactor would interact with all three DNA-binding proteins and then bridge them to the core transcription complex. Although such a cofactor may exist, our findings suggest a narrower and more specific role for ß-catenin, namely in promoting a specific interaction with SF1 that unmasks its transactivation potential, thereby permitting synergistic interaction with EGR1.

Although we emphasize the functional interaction between ß-catenin and SF1 in transducing the GnRH transcriptional signal in gonadotropes, there are numerous other targets of the coactivator that play important roles in pituitary development and cell type specification. For example, ß-catenin acts as cofactor for T-cell factor/lymphoid enhancer factor transcription factors in fetal pituitaries that regulate expression of Pitx2 (40). ß-Catenin has also been shown recently to act as a cofactor for Prop1 in regulating expression of Pit1 and Hesx1 that are required for specification of somatotropes, thyrotropes, and lactotropes in embryonic pituitaries (41). Additionally, transcripts for Lhb are substantially reduced in fetal mouse pituitaries that harbor targeted disruption of their ß-catenin alleles (41). Together these data suggest that ß-catenin can also act as an essential cofactor for multiple transcription factors that control pituitary cell type specification.

Although our study highlights the role for ß-catenin as a required component of the GnRH transcriptional signal, it is likely that this coactivator facilitates the transcriptional effect of other signaling cascades initiated by peptide hormones binding to G protein-coupled receptors (GPCRs). For instance, we have recently reported that FSH regulation of aromatase (Cyp19a1) transcription in granulosa cells also requires a selective interaction between ß-catenin and SF1 (42). GnRH is generally viewed to activate Lhb expression primarily via Gq/11 (43). In contrast, FSH signals via Gs (44). Consequently, the transcriptional effect of ß-catenin may not be limited to specific classes of GPCRs. Moreover, the presence of GPCRs/ß-catenin/SF1 transcriptional programs in pituitary and ovary suggests a broad and previously underappreciated role for this cofactor in the hypothalamic-pituitary-gonadal axis.

In summary, normal gonadal function requires continuous pulsatile secretion of both GnRH and LH (2, 4, 6). Sustained pulsatile secretion of LH exerts a transcriptional demand on the genes that encodes its subunits (37, 38, 39). Our results have exposed a new and critical role for ß-catenin where it functions as a coactivator of SF1, rendering the orphan receptor capable of functionally synergizing with GnRH-induced EGR1. The synergistic interdependency of ß-catenin, SF1, and EGR1 provides a means of amplifying the transcriptional response of LHB/Lhb genes to GnRH to ensure that sufficient amounts of LH are available for sustained pulsatile secretion, a requirement for fertility in both males and females.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
GnRH, phenylmethylsulfonylfluoride, Triton X-100, Igepal CA-300, salmon sperm, sodium deoxycholate, and Nonidet P-40 were purchased from Sigma Chemical Co. (St. Louis, MO); sodium dodecyl sulfate (SDS) was from Bio-Rad Laboratories (Hercules, CA); formaldehyde was from Fisher Scientific (Pittsburgh, PA); glycine was from J. T. Baker (Phillipsburg, NJ).

DNA Constructs and siRNA
The –779/+10-bp bovine LHB reporter construct has been described (9, 17, 45). The Egr1 expression vector (CMVNGF1A) was kindly provided by Dr. Jeffery Milbrandt (Washington University Medical School, St. Louis, MO) (46); the murine Axin1 in a pcS2+MT expression vector was generously provided by Dr. Frank Costantini [Columbia University, New York, NY (30)]; and the murine {Delta} 90 ß-catenin in the pUHD10–3 vector was provided by Dr. James Nelson (Stanford University of Medicine, Palo Alto, CA) (34). The pCMX-SF-1 and SF-1 235–4A expression vectors were kindly provided by Dr. Ken-Ichirou Moroshashi (University of Tsukuba, Tsukuba, Japan) (28). Plasmids encoding N terminally myc-tagged Sf1 or Sf1 235–4A were prepared by digesting pCMX-Sf1 or Sf1 235–4A with EcoRI (Invitrogen, Carlsbad, CA) and ligated into the EcoRI site of pCMVTag3B (Stratagene, La Jolla, CA).

Smart pools of interfering RNA for Catnb and nontargeting interfering RNA were obtained from Dharmacon.

Cell Culture and Transient Transfections
LßT2 cells were maintained at 37 C with 5% CO2 in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen Life Technologies). Before transfection, LßT2 cells were plated (24-well plates; 250,000/well) and maintained in DMEM supplemented with 10% FBS. After 24 h, cells were washed with PBS (Invitrogen Life Technologies) and a transfection cocktail containing DNA, lipofectamine (2 µl/well; Invitrogen Life Technologies) and DMEM was added. An equal volume of DMEM supplemented with 20% FBS was added 12–16 h later. Cells were assayed 24 h later for reporter gene activity (Dual Luciferase Reporter assay kit; Promega Corp., Madison, WI). The amount of DNA was maintained constant by adding an empty vector containing the CMV promoter.

For siRNA experiments with GnRH, a suspension of 250,000 LßT2 cells per well was transfected with lipofectamine (2 µl/well), siRNA, and DNA in DMEM, and cells were allowed to attach to a 24-well plate. Six hours post transfection, DMEM containing 20% FBS was added to each transfection well. Cells were treated 72 h post transfection with either vehicle or 10 nM GnRH for 24 h, and reporter activity was measured.

To examine the activity of EGR1 and SF1 in the presence of siRNA, preplated LßT2 cells (250,000 cells per well in a 24-well plate) were transfected (lipofectamine, 2 µl/well) with DNA and siRNA, and reporter activity was assayed 72 h post transfection.

Immunoprecipitation and Immunoblot Analysis
For immunoprecipitation experiments, LßT2 cells were plated into 150-mm plates in complete media. Once cells were 70% confluent they were dosed with either GnRH (10 nM) or vehicle (PBS) in complete media. Cells were washed 60 min post GnRH or vehicle with cold PBS, and crude nuclear lysate was isolated as described by Gummow et al. (27). Nuclear extracts (1 mg) were then incubated with 15 µg of agarose-conjugated IgG (sc-2346) or ß-catenin beads (sc-1496 AC) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 C while rotating. Protein complexes where collected after a brief centrifugation and washed three times with PBS. Complexes were boiled in sample buffer and assayed by immunoblot.

For immunoblot analysis of siRNA-mediated knockdown of ß-catenin, 4 µg of transfection lysate was subjected to SDS-PAGE and transferred to polyvinylidine difluoride membrane (Bio-Rad Laboratories). Membranes were then blocked for 1 h at room temperature in Tris-buffered saline solution containing 0.05% Tween 20 (TBST) and 5% nonfat dry milk. Membranes were then probed with either anti-ß-catenin (BD Transduction Laboratories, Lexington, KY; catalog no. 610153) antibody diluted 1:5000 or anti-AKT (Cell Signaling Technology, Beverly, MA) antibodies diluted 1:2000 in TBST containing 5% nonfat dry milk for either 1 h at room temperature or overnight at 4 C. Membranes were then rinsed twice for 5 min each in TBST and then incubated for 1 h at room temperature with either an antimouse or an antirabbit IgG secondary antibody conjugated to a horseradish peroxidase enzyme (Amersham Pharmacia Biotech, Arlington Heights, IL) diluted 1:40,000. The membrane was then rinsed three times in TBST and subjected to enhanced chemiluminescence (Amersham Pharmacia Biotech).

Immunoblot analysis of IP reactions were performed in a similar fashion, except that membranes were probed with either anti-SF1 [Upstate Biotechnology (Lake Placid, NY)/Millipore Corp. (Bedford, MA)] diluted 1:5000 or an anti ß-catenin (H-102; Santa Cruz Biotechnology) diluted 1:5000 in TBST containing 5% nonfat dry milk overnight at 4 C.

ChIP Assays
Ten million LßT2 cells were plated into 100-mm plates for 24 h, treated with vehicle or 10 nM GnRH as indicated above, and then cross-linked with formaldehyde at a final concentration of 1% for 10 min at room temperature. Glycine was then added to a final concentration of 125 mM. After 5 min, cells were scraped and centrifuged at 400 x g for 5 min at 4 C. Cell pellets were lysed with 1 ml of SDS lysis buffer (1% SDS; 10 mM EDTA; 50 mM Tris-HCl, pH 8) plus protease inhibitors (Complete mini Tabs; Roche Diagnostics, Indianapolis, IN) on ice for 10 min and sonicated (DNA length of ~500 kb). Chromatin (150 µl) per immunoprecipitation was diluted 1:10 in dilution buffer (16.7 mM Tris-HCl, pH 8; 167 mM NaCl; 1.2 mM EDTA; 0.01% SDS; 1.1% Triton X-100,) and incubated with 5 µg of anti-Egr-1 (C-19/C-588; Santa Cruz Biotechnology), anti-ß-catenin (H102; Santa Cruz Biotechnology), anti-SF1 (Upstate Biotechnology/Millipore), or control rabbit IgG (Santa Cruz Biotechnology) by rotation at 4 C overnight with 60 µl of Protein G Agarose (Upstate Biotechnology/Millipore). Protein G complexes were collected by centrifugation, and beads were rinsed one to three times with buffer A (20 mM Tris-HCl, pH 8; 150 mM NaCl; 2.0 mM EDTA; 0.1% SDS; 1.0% Triton X-100) and once with buffer B (same as buffer A except without 500 mM NaCl). Complexes were eluted with 450 µl of elution buffer (0.1 M NaHCO3, 1% SDS) for 30 min. Resulting supernatants were collected and NaCl (final concentration of 0.3 M) and proteinase K (20 µg/ml) were added to eluates and incubated at 65 C for 5 h. DNA was isolated using phenol chloroform extraction and resuspended in 30 µl of H2O. DNA (1 µl) was subjected to 30 cycles of standard PCR using primers designed to amplify a distal fragment 5'-cctccttggtgttggagaaa-3' and 5'-gagagtgggaggtggctaga-3' and a proximal fragment 5'-tcaccttctccttgggtgtc-3' and 5'-gtcctcccctgctgtgttta-3' of the Lhb gene.

Statistics
Reporter activity was analyzed by one-way ANOVA, and differences among treatments were determined with the Newmans-Keuls multiple comparison test (Figs. 2Go, 4Go, and 5Go). Reporter activity was analyzed by two-tailed Student’s t test in Figs. 1Go and 3Go. ChIP assays in Fig. 6Go were analyzed by a one-paired Student’s t test.


   ACKNOWLEDGMENTS
 
We thank Drs. Mary E. Hunzicker-Dunn, Jen Weck, and Ted Chauvin for critical evaluation of this manuscript and Drs. Lisa Gloss and Tehnaz Parakh for insightful discussion.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant R01-DK-28559 (to J.H.N) and a Fellowship award from the Lalor Foundation (to T.B.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 23, 2007

Abbreviations: ChIP, Chromatin immunoprecipitation; CMV, cytomegalovirus; EGR1, early growth response 1; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; PITX1, pituitary homeobox 1; SDS, sodium dodecyl sulfate; SF1, steroidogenic factor 1; TBST, Tris-buffered saline solution containing 0.05% Tween 20.

Received for publication September 11, 2006. Accepted for publication January 19, 2007.


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