Releasing Hormone

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Molecular Endocrinology 21(12):3028–3038 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2007-0268

Nuclear Stabilization of ␤-Catenin and Inactivation of Glycogen Synthase Kinase-3␤ by GonadotropinReleasing Hormone: Targeting Wnt Signaling in the Pituitary Gonadotrope Samantha Gardner, Stuart Maudsley, Robert P. Millar, and Adam J. Pawson Medical Research Council Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, Edinburgh EH16 4TJ, Scotland, United Kingdom The GnRH receptor is a G protein-coupled receptor (GPCR), and its ligand GnRH is the central regulator of the reproductive system. GnRH receptors are known to target a wide variety of signal transduction pathways. Several recent studies have shown that activation of GPCRs can impact on ␤-catenin signaling. ␤-Catenin is the main effecter of the Wnt signaling pathway where it acts with the transcription factors T cell factor/lymphoid enhancer factor to mediate the transcription of Wnt target genes. We show that GnRH treatment promotes the nuclear accumulation of ␤-catenin, activation of T cell factor-dependent transcription, and up-regulation of Wnt target genes, c-Jun, Fra-1, and c-Myc.

These results are observed in human embryonic kidney 293/GnRH receptor-expressing cells and have been recapitulated in L␤T2 and ␣T3-1 mouse gonadotrope cells. In addition to these findings, we show that GnRH treatment mediates the inactivation of glycogen synthase kinase-3, a protein serine/threonine kinase that regulates ␤-catenin degradation within the Wnt signaling pathway. Our findings extend the number of GPCRs that can target ␤-catenin signaling through diverse pathways. Furthermore, this is the first demonstration of the targeting of Wnt/␤-catenin signaling by a peptide hormone GPCR. (Molecular Endocrinology 21: 3028–3038, 2007)

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Stimulation of the Wnt pathway leads to the inhibition of GSK-3␤, by a poorly defined mechanism, and consequent stabilization of ␤-catenin levels. ␤-Catenin accumulates in the nucleus where it acts as a cofactor to TCF/LEF transcription factors to promote the transcription of Wnt target genes, many of which are key in developmental processes. Consequently, the dysregulation of ␤-catenin signaling has been reported to impact on the development of a number of cancers (7, 8). The hypothalamic decapeptide GnRH is the central regulator of the reproductive system through the stimulation of gonadotropin (LH and FSH) synthesis and secretion from the pituitary (9). These processes are mediated by the type I GnRH receptor, a GPCR present on pituitary gonadotropes, that signals predominantly via the heterotrimeric Gq protein leading to the elevation of intracellular Ca2⫹ levels, activation of the protein kinase C (PKC)/MAPK signaling cascade, and regulation of gonadotropin gene transcription and secretion (10, 11). GnRH analogs are extensively used in the treatment of hormone-dependent diseases and are the major therapeutics of prostate cancers, uterine fibroids, and endometriosis. Given the important clinical applications of GnRH analogs, and the crucial roles of GnRH in regulating reproductive axis development and function, it is of considerable interest to identify and understand the full spectrum of signal transduction pathways that are targeted by the type I GnRH re-

VER THE PAST 6 yr an intriguing connection between classical G protein-coupled receptor (GPCR) signaling and the targeting of ␤-catenin activity has begun to emerge after the seminal demonstration of prostaglandin F2␣ stimulation of ␤-catenin nuclear transcriptional activity via the FPB prostanoid GPCR (1–3). ␤-Catenin has a well-established role in both cell-cell adhesion, where it acts as a central adaptor protein linking cadherins to the actin cytoskeleton at adherens junctions, and as a signaling molecule in the Wnt/␤-catenin developmental signaling pathway, where it acts as a cofactor with the transcription factors T-cell factor (TCF)/lymphoid enhancer factor (LEF) in mediating the transcription of Wnt target genes (4–6). In the absence of Wnt ligand stimulation, the cellular levels of ␤-catenin are kept low by a destruction complex that includes adenomatous polyposis coli and axin, bound by casein kinase I and glycogen synthase kinase-3␤ (GSK-3␤), which phosphorylate ␤-catenin and target it for degradation. First Published Online August 23, 2007 Abbreviations: DAPI, 4⬘,6-Diamidino-2-phenylindole; DPBS, Dulbecco’s PBS; GPCR, G protein-coupled receptor; GSK-3␤, glycogen synthase kinase-3␤; HEK, human embryonic kidney; LEF, lymphoid enhancer factor; NS, nonstimulated; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; TCF, T-cell factor. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

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ceptor, both at the level of the pituitary and in normal and malignant peripheral tissues (12–14). We now show that GnRH agonists can induce the nuclear accumulation of ␤-catenin, activation of TCFdependent transcription, and up-regulation of Wnt target genes, in addition to inhibition of GSK-3, in a model human embryonic kidney (HEK)293 cell line expressing the type I GnRH receptor and in the L␤T2 gonadotrope cell line. Our findings provide the first demonstration of the targeting of Wnt/␤-catenin signaling by a peptide hormone GPCR.

was not statistically different. GnRH I-induced ␤-catenin nuclear accumulation was inhibited by cotreatment with a type I GnRH receptor antagonist (GnRH-Ant) (Fig. 1C), providing evidence for the observed effects being specifically mediated by GnRH binding and activation of the type I GnRH receptor. The L␤T2 gonadotrope cell line exhibits the characteristics of fully differentiated pituitary gonadotropes and is a relevant model to study the mechanisms of pituitary gonadotrope cell function (15, 16). Accordingly, we assessed whether our observations in the HEK293 model cell line could be extended to the gonadotrope. Indeed, we observed a statistically significant nuclear accumulation of ␤-catenin in L␤T2 gonadotrope cells in response to a 30-min stimulation with GnRH I (Fig. 1D).

RESULTS For all results, we first describe findings in the HEK293 model cell line expressing the type I GnRH receptor and then demonstrate the effects in the physiologically relevant L␤T2 gonadotrope cell line. GnRH Induces ␤-Catenin Nuclear Accumulation A time course of GnRH I treatment demonstrates ␤-catenin accumulation in nuclear extracts obtained from HEK293/GnRH receptor-expressing cells, reaching maximal accumulation after 30 min (Fig. 1A). ␤-Catenin nuclear accumulation occurred in a dose-dependent manner in response to both endogenous forms of GnRH (GnRH I and GnRH II) (Fig. 1B). GnRH I appeared more potent than GnRH II, but this

␤-Catenin Cellular Redistribution in Response to GnRH Immunofluorescence confocal microscopy was used to visualize ␤-catenin localization in the HEK293 and L␤T2 gonadotrope cells. Consistent with our previously published data, we observed cytoskeletal reorganization and changes in cell morphology in response to GnRH treatment in the HEK293 cells (17), in addition to a redistribution of ␤-catenin (data not shown). In the L␤T2 gonadotrope cells, ␤-catenin staining was predominately restricted to the plasma membrane in nonstimulated (NS) cells (Fig. 2), and we

Fig. 1. Immunoblots of GnRH-Induced ␤-Catenin Nuclear Accumulation in HEK293 and L␤T2 Cells A, Cells were treated with 1 ␮M GnRH I for the indicated times, or with vehicle control (20% propylene glycol) (NS). B, Cells were treated with decreasing doses of either GnRH I or GnRH II (in order of loading; NS, 10 ␮M, 1 ␮M, 100 nM, 10 nM, 1 nM) for 30 min. C, Cells were treated for 30 min with 1 ␮M GnRH I, 1 ␮M GnRH-Ant (a type I GnRH receptor antagonist), or both, or left untreated as indicated. D, L␤T2 cells were treated with 1 ␮M GnRH I for 30 min or with vehicle control. All panels show nuclear accumulated ␤-catenin. Representative blots are shown. Data from at least three independent experiments were quantified (using corresponding ERK2 or ␤-actin immunoblot as a loading control) and the mean fold over control ⫾ SE presented below the corresponding blot. Values of P ⬍ 0.05 are represented by an asterisk and represent statistical significance from NS or as otherwise shown. IB, Immunoblot; GnRH-Ant., GnRH antagonist; w.c. lysate, whole-cell lysate.

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Fig. 2. Immunofluorescence Confocal Microscopy of GnRH I-Induced ␤-Catenin Nuclear Accumulation L␤T2 cells were plated on poly-L-lysine treated eight-well chamber slides and serum starved (16 h) before stimulation. After stimulation with GnRH I for 30 min (B) or vehicle (NS) (A), cells were fixed and immunocytochemistry was performed. Detection of the immunoreactive proteins was achieved using Alexafluor (488 nM, green) conjugate for ␤-catenin. The merge also shows DAPI staining (red) showing colocalization as yellow. Higher magnification images are shown inset into the merge.

observed a marked stabilization of ␤-catenin levels, and colocalization of ␤-catenin with the nucleus after GnRH treatment (Fig. 2). These results are consistent with the events that follow activation of the ␤-catenin/ TCF signaling axis leading to transcription of Wnt target genes (4–6). GnRH Promotes the Activation of a TCF-Luciferase Reporter To investigate whether GnRH-induced ␤-catenin nuclear accumulation resulted in the activation of TCF/ LEF-dependent transcriptional activity, we transfected a TCF-luciferase reporter construct into HEK293 cells, L␤T2, and ␣T3-1 gonadotrope cells. After a 24-h ligand treatment, cells were assayed for luciferase activity. GnRH I stimulated a 3.5-fold induction of TCFdependent transcription of luciferase in HEK293 cells (Fig. 3A). In L␤T2 and ␣T3-1 gonadotrope cells, GnRH I treatment resulted in a 15- and 29-fold induction, respectively (Fig. 3, B and C). This induction was significantly inhibited by cotreatment with GnRH-Ant and a Gq inhibitor (Fig. 3).

GnRH Up-Regulates Wnt Target Genes To establish that GnRH activation of the TCF-luciferase reporter was functional in our model system, we employed a quantitative RT-PCR technique using Fra-1, c-Jun, and c-Myc as examples of Wnt target genes. In the HEK293 model cells a maximal 26-fold increase in Fra-1 mRNA levels at 6 h of stimulation with GnRH was observed (Fig. 4A). Time-dependent responses to GnRH were recorded for c-Jun revealing significant up-regulation after 30 min but decreasing again at 2 h (Fig. 4B). GnRH promotes a small but significant up-regulation of c-Myc in the HEK293 model at 1 h (Fig. 4C). Using the L␤T2 cell line to demonstrate up-regulation of Fra-1, c-Jun, and c-Myc, we confirm significant increases, at the same time points as the HEK293 model cell line, with maximal inductions of 29-, 28-, and 2.7-fold, respectively (Fig. 4, D–F). GnRH Phospho-Inhibits GSK-3 GSK-3 is a well-established negative regulator of Wnt signaling. In the inactive Wnt signaling pathway GSK-3

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receptor, when compared with GnRH II (EC50, 79 nM) (Fig. 5B). These potencies are consistent with our previously published measurements of inositol phosphate production in response to GnRH (11, 18) and provide evidence that GnRH-induced GSK-3␤ Ser9 phosphorylation occurs via signaling at the type I GnRH receptor. In the presence of GnRH-Ant the 4-fold increase in Ser9 phosphorylation was decreased to basal levels (Fig. 5C). Intriguingly, we observed no significant change in the Ser9 phosphorylation status of GSK-3␤ in the L␤T2 gonadotrope cells in response to GnRH I treatment (Fig. 5D). Mediators of GnRH-Induced Signaling to ␤-Catenin/TCF-Dependent Transcription

Fig. 3. GnRH Stimulation of TCF/␤-Catenin Nuclear Transcriptional Activity HEK293 (panel A), L␤T2 (panel B), and ␣T3–1 (panel C) cells were seeded into 12-well plates and transfected with the TOPflash TCF-luciferase reporter plasmid. Cells were serum starved 24 h after transfection for a further 16 h, followed by either vehicle control (NS), 1 ␮M GnRH I stimulation, 1 ␮M GnRH plus 1 ␮M GnRH-Ant, or 1 ␮M GnRH plus 100 nM Gq inhibitor, as indicated, for 24 h. Luciferase activity was expressed in arbitrary units relative to the activity observed in vehicle control cells normalized for Renilla luciferase activity (NS). This experiment was performed at least three times. Values of P ⬍ 0.05 are represented by an asterisk and represent statistical significance from NS unless dotted bars show otherwise. GnRH Ant, GnRH antagonist.

phosphorylates ␤-catenin, allowing its ubiquitination and subsequent degradation. After activation of Wnt signaling, GSK-3 is inhibited by a poorly defined mechanism, thereby allowing the stabilization and accumulation of ␤-catenin levels (an event that we have already observed in the present study; see Fig. 1). In classical insulin signaling, Akt/PKB phosphorylation of GSK-3␤ at Ser9 leads to its inhibition. We investigated whether GnRH signaling targets this inhibitory phosphorylation site. We observed the phosphorylation of GSK-3␤ at Ser9 in HEK293 model cells after a 5-min exposure to GnRH I (Fig. 5A). The response was transient and decreased toward basal levels after 30 min GnRH stimulation. GnRH treatment also inhibits GSK-3␣ by phosphorylation at Ser21 (data not shown). Dose-response Western blots demonstrate the higher potency of GnRH I (EC50, 4.6 nM), at the type I GnRH

To elucidate the signaling events leading to GnRH-induced activation of TCF-dependent transcriptional activity, we used the TCF-luciferase reporter assay system in our HEK293 model and L␤T2 gonadotrope cell lines. Cotransfection of a dominant-negative TCF4 construct resulted in a significant reduction in the GnRH-induced luciferase activity compared with vector control in both cell types (Fig. 6, A and B). Wild-type GSK-3␤ (GSK3WT) and constitutively active Ala9 GSK-3␤ mutant (GSK3S9A) constructs were employed to test whether GnRH-induced phospho-inhibition of GSK-3␤ at Ser9 was involved in the recruitment of ␤-catenin/TCF signaling. Cotransfection of the GSK-3␤ wild-type construct resulted in reduced GnRH-induced TCF-luciferase activity compared with vector control in both cell types (Fig. 6, C and D). This reduction was most likely due to the increased cellular pool of GSK-3␤ that is able to drive the degradation of ␤-catenin. Interestingly, GnRH-mediated activation of TCF-dependent transcription, in the presence of cotransfected GSK3WT or GSK3S9A mutant, was reduced to similar levels. This result suggests the phospho-inhibition of GSK-3␤ at Ser9 is not necessary for ␤-catenin/TCF signaling. The phosphatidylinositol-3kinase (PI3K)/Akt signaling axis is classically associated with GSK-3␣/␤ phospho-inhibition at Ser9/21. We assessed the potential role of this signaling pathway in mediating ␤-catenin/TCF signaling in response to GnRH. Treatment with the PI3K inhibitors, Wortmannin and LY294002, resulted in no significant alteration in GnRHinduced TCF-luciferase activation (Fig. 6, E and F). Our data suggest that GnRH-induced GSK-3␤ Ser9 phosphorylation is not involved in the GnRH-induced ␤-catenin nuclear stabilization and TCF-dependent transcription and excludes PI3K/Akt-mediated signaling in these events. Activation and Accumulation of c-Jun Protein in Response to GnRH Stimulation To further investigate the relationship between GnRH targeting ␤-catenin/TCF transcriptional activity and GSK-3␤ phospho-inhibition, and c-jun up-regulation, we analyzed the phosphorylation status of c-Jun by Western blot. A significant accumulation of c-Jun was

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Fig. 4. Effect of GnRH on Fra-1, c-Jun, and c-Myc mRNA Expression in HEK293 and LBT2 Cell Lines Real-time RT-PCR analysis of mRNA expression in HEK293 (A, B, and C) and L␤T2 (D, E, and F) treated with 1 ␮M GnRH I or vehicle (NS). Data are shown as mean fold over NS for each time point ⫾ SEM from at least three independent experiments; *, Significantly different values from NS (P ⬍ 0.05).

observed in the cytoplasmic fraction after 45 min of GnRH stimulation (Fig. 7, A and C). A nuclear fraction was collected, and a significant increase in c-Jun was present after 90 min of GnRH treatment (Fig. 7, B and C). Activation of c-Jun transcriptional activity requires its phosphorylation at Ser63 and Ser73 (19, 20). The N-terminal amino acid Ser73 in c-Jun was significantly phosphorylated in response to GnRH treatment (Fig. 7, B and C). c-Jun has been reported to be phosphorylated at Thr239 by GSK-3␤, and it has been further postulated that this site becomes dephosphorylated when GSK-3␤ is phospho-inhibited at Ser9 (21). We observed a 2-fold increase in Thr239 phosphorylation after 90 min treatment with GnRH (Fig. 7, B andC). However, no significant dephosphorylation of Thr239 was observed in our system. A marked nuclear accumulation of pSer73 c-Jun was observed by immunocytochemistry in the L␤T2 gonadotrope cells after 60 min stimulation with GnRH I (Fig. 7D).

DISCUSSION GnRH is the central regulator of the reproductive system through its stimulation of gonadotropin (LH and

FSH) synthesis and secretion from the pituitary (9). The type I GnRH receptor, a GPCR present on pituitary gonadotropes, signals predominantly via the heterotrimeric Gq protein leading to the elevation of intracellular Ca2⫹ levels, activation of the PKC/MAPK signaling cascade, and regulation of gonadotropin gene transcription and secretion, which control ovulation and spermatogenesis (10, 11). The mechanisms mediating the synthesis and secretion of LH and FSH have been studied in depth but are not yet fully understood. Recently, an important study highlighting the role of ␤-catenin as a member of a transcription factor complex that drives maximal activity of the LH␤ promoter in response to GnRH was published by Salisbury et al. (22). This work demonstrates the colocalization of ␤-catenin with steroidogenic factor 1 and early growth response 1 on the promoter of the LH␤-subunit gene in response to GnRH and suggests that endogenous steroidogenic factor 1 and ␤-catenin can physically associate in L␤T2 cells. Stabilization and nuclear accumulation of ␤-catenin are pre-eminent hallmarks of the Wnt/␤-catenin signaling pathway. The seminal demonstration of GPCR activation of ␤-catenin/TCF-mediated transcriptional activity was reported for the prostanoid FPB receptor

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Fig. 5. Immunoblots of GnRH-Induced GSK-3␤ Phosphorylation in HEK293 and L␤T2 Cells A, HEK293 cells were treated with 1 ␮M GnRH I for the indicated times or with vehicle control (NS). B, HEK293 cells were treated with increasing doses of either GnRH I or GnRH II (in order of loading; NS, 1 nM, 10 nM, 100 nM, 1 ␮M, 10 ␮M) for 30 min. C, HEK293 cells were treated for 30 min with 1 ␮M GnRH I, 1 ␮M GnRH-Ant, or both, or vehicle control as indicated. D, L␤T2 cells were treated with 1 ␮M GnRH I for the indicated times, or with vehicle control (NS). All panels show cytoplasmic GSK-3␤. Representative blots are shown. Data from at least three independent experiments were quantified (using corresponding ERK immunoblots as a loading control) and the mean fold over control ⫾ SE is presented below the corresponding blot. *, Significantly different values from NS or as otherwise shown by dotted lines (P ⬍ 0.05). In panel B, the asterisk represents statistical significance of GnRH I from GnRH II at 10 nM. IB, Immunoblot; GnRH-Ant, GnRH antagonist; w.c. lysate, whole-cell lysate.

and suggested that this may be a property of other GPCRs (1, 23). Here we have shown that ␤-catenin accumulates in the nucleus, activates a TCF-luciferase reporter, and promotes up-regulation of Wnt target genes in response to GnRH in gonadotrope cells. Thus, ␤-catenin signaling in response to GnRH stimulation in L␤T2 gonadotrope cells is not only important as a cofactor for TCF/LEF-dependent transcriptional activity at Wnt target genes, as demonstrated in the present study, but also has an important role in mediating gonadotropin gene expression (22). Furthermore, our demonstration that GnRH stimulates

␤-catenin/TCF signaling in an heterologous cell system (i.e. the HEK293 model cell line) has implications for GnRH impacting on Wnt/␤-catenin signaling processes in a variety of peripheral tissues and cancers that express the type I GnRH receptor. ␤-Catenin has a well-established role in both cellcell adhesion, where it acts as a central adaptor protein linking cadherins to the actin cytoskeleton at adherens junctions. A number of studies have suggested that the sequestration of ␤-catenin into the cadherin complex, by signals that promote strong cell-cell adhesion, would inhibit TCF-dependent transcription,

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Fig. 6. Key Mediators Involved in GnRH Stimulation of TCF/␤-Catenin Nuclear Transcriptional Activity HEK293 cells were either: cotransfected with equal amounts of the TOPflash (TCF reporter plasmid) and a dominant-negative TCF4 construct or vector control (A); TOPflash and a wild-type GSK-3␤ (GSK3␤-WT), Mutant GSK-3␤ (GSK3␤-S9A) or vector control (C). L␤T2 cells were either cotransfected with equal amounts of the TOPflash (TCF reporter plasmid) and a dominantnegative TCF4 construct or vector control (B); TOPflash and a wild-type GSK-3␤ (GSK3␤-WT), mutant GSK-3␤ (GSK3␤-S9A) or vector control (D). All cells were serum starved 24 h after transfection for a further 16 h, followed by either vehicle control (NS), 1 ␮M GnRH stimulation, or Wortmannin (100 nM) (E), or LY294002 (5 ␮M) (F) as indicated for 24 h. Luciferase activity was expressed in arbitrary units relative to the activity observed in vehicle control (NS)-treated cells normalized for Renilla luciferase activity. Values of P ⬍ 0.05 are represented by an asterisk and represent statistical significance from NS unless otherwise shown by dotted lines.

whereas signals that destabilize cadherin-catenin complexes at adherens junctions, and release ␤-catenin from these sites for translocation to the nucleus, would enhance TCF/␤-catenin-dependent transcription of TCF target genes (5, 24, 25). These studies suggest that ␤-catenin can be mobilized and redistributed to the nucleus to affect TCF-dependent transcription. The cytoskeletal reorganization and changes in cell morphology observed in GnRH-treated cells (Ref. 17 and present study), with a corresponding ␤-catenin redistribution and accumulation in the nucleus in our study, accords with this scenario of events. Initially the GSK-3␤ Ser9 phosphorylation event appeared to be connected with the observed ␤-catenin accumulation in response to GnRH, based on the well-

established role of GSK-3 as a negative regulator of Wnt signaling as reported in the literature. However, recent mouse knock-in analysis studies performed by Alessi and colleagues (26) suggested GSK-3␤ Ser9 phosphorylation is not involved in Wnt signaling. Indeed, in the present study, GnRH-induced TCF-dependent transcription was similar both in the presence of cotransfected GSK3WT or GSK3S9A, further suggesting that Ser9 phospho-inhibition of GSK-3 is not a requirement for ␤-catenin/TCF-dependent transcription. In L␤T2 cells we observed high basal levels of pSer9 GSK-3␤ by immunoblot analysis, and no increase in phospho-inhibition in response to GnRH was evident. This result appears to agree with the findings that Ser9 phosphorylation is not necessary for ␤-cate-

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Fig. 7. Phosphorylation Status of c-Jun in Response to Stimulation by GnRH A, HEK293 cells were treated with 1 ␮M GnRH-I for the indicated times, or with vehicle control (NS). The cytoplasmic fraction was then immunoblotted as normal. B, The samples from panel A were first subjected to a slow-speed spin to pellet the nuclei, and the nuclear fraction was separated from the cytoplasmic and then sonicated to lyse the nuclear membrane. The nuclear fraction was then immunoblotted as normal. Representative blots are shown. C, Quantification of the data from at least three independent experiments (using corresponding ERK immunoblots as a loading control) and the mean fold over control ⫾ SE is presented. *, Significantly different values from NS (P ⬍ 0.05). D, Immunocytochemistry of L␤T2 cells after 60 min stimulation with 1 ␮M GnRH I. Detection of the immunoreactive proteins was achieved using Alexafluor (546 nM, green) conjugate for c-Jun (Ser73). The merge also shows DAPI staining (red) showing colocalization as yellow. IB, Immunoblot; w.c. lysate, whole-cell lysate.

nin/TCF-dependent transcription (Fig. 5D). Alternatively, the high basal levels may have prevented us from detecting any change in Ser9 phosphorylation status. However, in our nonpituitary cell model, the HEK293 cell line, GnRH-induced Ser9 phosphorylation in response to GnRH was clearly apparent, leaving us with the intriguing possibility that peripheral cells that express the type I GnRH receptor may recruit alternative signaling pathways.

GSK-3 is a multifunctional kinase with a diverse array of cellular targets influencing processes such as insulin signaling and cell fate decisions (27). GnRH-mediated phospho-inhibition of GSK-3 provides scope for further investigation of the impact of GnRH on these processes. One such target of GSK-3 is the Wnt-regulated transcription factor, c-Jun, which we demonstrated here to be up-regulated in response to GnRH (Fig. 4). It has been previously demonstrated that GSK-3 can phosphorylate

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c-Jun protein in vitro at Thr239, Ser243, and Ser249, to decrease DNA binding, (28) and a recent reinvestigation of c-Jun phosphorylation sites states that Thr239 phosphorylation is catalyzed by GSK-3 (21). Our investigation has shown that GnRH can cause the phospho-inhibition of GSK-3␤ and this, we postulated, would cause a dephosphorylation event at Thr239. However, we could not detect a dephosphorylation in response to GnRH, with the method used, and instead observed an increase in pThr239 after 90 min stimulation. Possible explanations for the increased pThr239 are: a reduced GnRH-induced GSK-3␤ phospho-inhibition at 90 min, thereby allowing GSK-3 to rephosphorylate Thr239; or increased detection of pThr239 due to the increase in total c-Jun protein in the nucleus. The lack of GnRH-induced dephosphorylation at Thr239 is perhaps not surprising in light of the findings of Morton et al. (21), which demonstrated that lipopolysaccharide- or anisomycin-induced dephosphorylation of c-Jun at Thr239 does not require the inhibition of GSK-3. The cross talk between the c-Jun/AP-1 and Wnt signaling pathways has been demonstrated in several previous studies (29–31). The up-regulation of c-Jun in response to GnRH highlights the potential cross talk between AP-1 and Wnt signaling. This study implicates GnRH as a regulator of c-Jun and suggests, due to the ability of c-Jun to positively regulate itself (32) and to bind to the TCF-4 transcription factor (33), that GnRH can activate ␤-catenin/TCF signaling through c-Jun. The studies by Regan and colleagues on prostaglandin F2␣ activation of ␤-catenin/TCF nuclear signaling via the FPB receptor provide a valuable comparison for our studies on GnRH signaling (1). Both studies employed the intracellular context of HEK293 cells, and both receptors couple predominantly to Gq-signaling and phospholipase C ␤ activation. Furthermore, both receptors have a truncation of the carboxyl-terminal tail resulting in an absence of rapid desensitization and prolonged ligand-induced signaling (34, 35). Both receptors also exhibit ligand-independent constitutive internalization (Pawson, A. J., manuscript in preparation; and Refs. 36 and 37). Activation of small GTPases to induce changes in cell morphology have been demonstrated for both the FPB and GnRH receptors (1, 17, 38). Interestingly, activation of ␤-catenin/TCF signaling by the FPB receptor was shown to occur by PKC/Rho-mediated changes in cell morphology (38). It will be interesting to determine whether the similarities outlined above are a general requirement for GPCR targeting of ␤-catenin/TCF nuclear signaling. Our results have important implications for ␤-catenin/TCF signaling in regulating pituitary gonadotrope cell function and point to a more general role for GnRH regulation of Wnt target gene expression in a variety of normal physiological and pathophysiological reproductive cell types and tissues. Furthermore, our findings with the type I GnRH receptor, together with the studies on the prostanoid (1, 23), acetylcholine muscarinic (2), lysophosphatidic acid (3), and thromboxane receptors (39), suggest that activation of Wnt/␤-

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catenin signaling through diverse pathways may be further extended to other GPCRs.

MATERIALS AND METHODS Plasmids The wild-type and Ser9 GSK-3␤ mutants were a kind gift from D. Alessi, MRC Protein Phosphorylation Unit. The dominantnegative TCF4 mutant construct was a generous donation from E. Fearon, University of Michigan. Materials The PI3K inhibitors LY294002 (5 ␮M) and Wortmannin (100 nM) were all obtained from Calbiochem (La Jolla, CA). GnRH I, GnRH II, and the vehicle control (20% propylene glycol) were obtained from Sigma (St. Louis, MO). GnRH-Ant was a kind gift from J. Rivier. The Gq inhibitor was a generous donation from M. Taniguchi (Astellas Pharma, Inc., Tokyo, Japan). Cell Culture HEK293 cells stably expressing the rat type I GnRH receptor generated within our laboratory (40), L␤T2 cells, and ␣T3–1 (obtained from P. Mellon, University of California) were maintained as previously described (12, 17). Before stimulation appropriately transfected or untransfected cells were incubated in serum-free media (DMEM, 2% glutamine, 1% penicillin/streptomycin, 10 mM HEPES) for 4 h for Western blotting experiments and 16 h for luciferase assays. Agonist stimulations were performed at 37 C in serum-free media after preincubation with chemical inhibitors, as necessary, as described in the figure legends. Preparation of Crude Nuclear Extracts and Immunoblotting After ligand stimulation, cell monolayers were lysed as previously described (17). Cell nuclei were crudely extracted from solubilized lysates by a 400 ⫻ g low-speed spin, and nuclei contents were released by sonication. The remaining cytoplasmic lysate was clarified by centrifugation at 20,000 ⫻ g for 10 min. Immunoblotting was performed using a 1:1000 dilution of mouse antihuman ␤-catenin (E-5), goat ␤-actin (I-19), and rabbit ERK2 (C-14) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Phospho-c-Jun(Thr239) (LabVision Corp., Fremont, CA) and Phospho-GSK-3␤(Ser 9), c-Jun, Phospho-c-Jun(Ser73), and ERK1/2 (Cell Signaling Technology, Beverly, MA). Visualization of the proteins was achieved by addition of a 1:1000 dilution of alkaline phosphataseconjugated polyclonal IgG-labeled protein (Sigma), using an enzyme-linked chemifluorescence reaction (Amersham Pharmacia Biotech, Piscataway, NJ) and quantified using a Typhoon 9400 Phosphoimager and ImageQuant TL software (Amersham Biosciences, Buckinghamshire, UK). Immunocytochemistry and Confocal Laser Microscopy L␤T2 cells were plated on poly-L-lysine-treated eight-well chamber slides (Nunc-Nalgene, Naperville, IL) at a density of 200,000 cells per chamber and serum starved (16 h) before cellular stimulation. After stimulation with 1 ␮M GnRH I for the indicated times, cell monolayers were washed twice with ice-cold Dulbecco’s PBS (DPBS) (with Ca2⫹/Mg2⫹) and then fixed in 200 ␮l of 100% methanol (MeOH) at ⫺20 C for 10 min. Following fixing the monolayers were washed in DPBS and incubated for 30 min in a Nonidet P-40-based cell permeabilization solution (DPBS, 10% fetal calf serum, 1% BSA, 0.2% Nonidet P-40) at room temperature. After permeabili-

Gardner et al. • GnRH Activation of Wnt/␤-Catenin Signaling

zation the fixed cells were blocked in a DPBS-based blocking solution (DPBS, 10% fetal calf serum, 1% BSA) for 1 h at room temperature or 16 h at 4 C. To visualize, the permeabilized cells were incubated for 16 h at 4 C with antisera at a dilution of 1:100. Detection of the specifically immunoreactive proteins was achieved by incubation with a 1:200 dilution of an Alexafluor (Molecular Probes; Invitrogen, Carlsbad, CA) (488 nM or 546 nM) antimouse or antirabbit conjugate for 1 h at room temperature. After secondary antibody removal, cell monolayers were subjected to 4⬘,6-diamidino2-phenylindole (DAPI) (Sigma) staining (1:2000) for 5 min and then washed three times in DPBS and mounted in Permafluor fixative (Immunotech, Marseilles, France). Confocal laser microscopy was performed on a Zeiss LSM510 laser scanning microscope (Carl Zeiss, Thornwood, NY).

Mol Endocrinol, December 2007, 21(12):3028–3038 3037

Statistical Analyses All experiments were repeated at least three times on independent days. Transient transfection experiments were also performed in triplicate wells each day. Statistical significance was set at P ⬍ 0.05, indicated by asterisks in figures, and analyses were performed using Student’s t test.

Acknowledgments We thank Pamela Brown and Nancy Nelson from the Biomolecular Core Facility for their assistance with the dual luciferase assays and Kevin Morgan, Elena Faccenda, and Sharon Battersby for their help with the PCR. The Gq inhibitor was a generous donation from M. Taniguchi (Astellas Pharma, Inc.)

TCF-Luciferase Reporter Gene Assay Transient transfections were performed using Superfect Reagent according to manufacturer’s instructions (QIAGEN, Chatsworth, CA) for the HEK293 cells. Fugene 6 Reagent was used according to the manufacturer’s instructions (Roche Clinical Laboratories, Indianapolis, IN) for the gonadotrope cell lines. Cells were seeded into 12-well plates and the next day transfected with TOPflash (Upstate Biotechnology, Inc., Lake Placid, NY), or cotransfected with various mutant constructs or vector control where appropriate, as well as a Renilla luciferase construct to control for transfection efficiency (Promega Corp., Madison, WI). Luciferase activity was assayed using a Dual-light Luciferase assay kit (Promega) in a FLUOStar Optima luminometer (BMG Lab Technologies, Buckinghamshire, UK). Luciferase activity was expressed in arbitrary units relative to the activity observed in unstimulated control cells normalized for Renilla luciferase activity. RT-PCR Cells were plated at 2 ⫻ 106 per 6-cm dish and left for 24 h before serum starving for a further 16 h. After ligand stimulation for a desired time-point the media were removed, and RNA was extracted from cells using Total RNA Isolation Reagent (TRIR; Abgene, Epsom, UK) according to the manufacturer’s instructions. Quantified RNA samples were reverse transcribed, and quantitative RT-PCR was performed as described previously using the Taqman ABI Fast 7900HT (41). Primer sequences were as follows. Mouse c-myc: [forward (F)], 5⬘-AGCCCCTAGTGCTGCATGAG-3⬘; [reverse (R)], 5⬘CCACAGACACCACATCAATTTCTT-3⬘; and Probe, 5⬘FAM-CCCACCACCAGCAGCGACTCTGA-TAM-3⬘; human c-myc: F, 5⬘-TGAGGAGACACCGCCCAC-3⬘; R, 5⬘CAACATCGATTTCTTCCTCATCTTC-3⬘; and Probe, 5⬘FAM-CCAGCAGCGACTCTGAGGAGGAACA-TAM-3⬘; mouse/human c-jun: F, 5⬘-GGATCAAGGCGGAGAGGAA3⬘; R, 5⬘-TTCCTTTTTCGGCACTTGGA-3⬘; and Probe, 5⬘FAM-CGCATGAGGAACCGCATCGCT-TAMRA-3⬘; human Fra-1: F, 5⬘-GGAGGAAGGAACTGACCGACTT-3⬘; R, 5⬘TGCAGCCCAGATTTCTCATCT-3⬘; and Probe, 5⬘-FAMCCAGTTTGTCAGTCTCCGCCTGCAG-TAMRA-3⬘; and mouse Fra-1: F, 5⬘-AACCGGAAGCACTGCATACC-3⬘; R, 5⬘-AAAACCAGACTCGGAGTAAAAGGA-3⬘; and Probe, 5⬘FAM-CCACGCTCATGACCACACCCTCTCT-TAMRA-3⬘. The ribosomal 18S primers and probe sequences are as follows: F, 5⬘-CGGCTACCACATCCAAGGAA-3⬘; R, 5⬘GCTGGAATTACCGCGGCT-3⬘; and probe (VIC labeled), 5⬘-TGCTGGCACCAGACTTGCCCTC-3⬘ (41). The expression of each gene was normalized for RNA loading for each sample using the 18S rRNA as an internal standard. Results are expressed relative to a sample of cDNA from normal endometrium included in every PCR as an internal standard. Fold increase in gene expression was determined by dividing the relative expression of that gene in GnRH-treated samples by the level of expression in vehicle-treated samples at the same time points.

Received May 23, 2007. Accepted August 13, 2007. Address all correspondence and requests for reprints to: Adam J. Pawson, Medical Research Council Human Reproductive Sciences Unit, The Queen’s Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, Scotland, United Kingdom. E-mail: [email protected]. Results from this work were presented in part at the 87th Annual Meeting of The Endocrine Society, San Diego, California, 2005 (Abstract P1-166, p. 209). Disclosure Statement: S.G., S.M., and A.J.P. have nothing to disclose. R.P.M. consults for Ardana plc.

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