Phosphorylation of the Hinge Domain of the Nuclear Hormone Receptor LRH-1 Stimulates Transactivation*

The nuclear receptor LRH-1 (NR5A2) functions to regulate expression of a number of genes associated with bile acid homeostasis and other liver functions, but mechanisms that modulate its activity remain unclear. We have found that mitogenic stimuli, including treatment with phorbol myristate (PMA), increase LRH-1 transactivation. This response maps to the hinge and ligand binding domains of LRH-1 and is blocked by the mitogen-activated protein kinase ERK1/2 inhibitor U0126. LRH-1 is a phosphoprotein and hinge domain serine residues at 238 and 243 are required for effective phosphorylation, both in vitro and in cells. Preventing phosphorylation of these residues by mutating both to alanine decreases PMA-dependent LRH-1 transactivation and mimicking phosphorylation by mutation to positively charged aspartate residues increases basal transactivation. Although serine phosphorylation of the hinge of SF-1 (NR5A1), the closest relative of LRH-1, confers a similar response, the specific targets differ in the two closely related orphan receptors. These results define a novel pathway for the modulation of LRH-1 transactivation and identify specific LRH-1 residues as downstream targets of mitogenic stimuli. This pathway may contribute to recently described proliferative functions of LRH-1.

The nuclear hormone receptor LRH-1 (NR5A2) is the mammalian homolog of the Drosophila transcription factor Fushi Tarazu-Factor1, which regulates Drosophila metamorphosis (1,2). A mouse protein termed LRH-1 3 (liver receptor homolog-1) was the first isolated mammalian orthologue 4 and further analysis of rat and human homologs revealed several isoforms (4 -7). Unlike the majority of nuclear receptors that function as dimers, LRH-1 binds as a monomer to sites found in a number of target genes that consist of a consensus nuclear receptor half-site with an upstream extension: 5Ј-(T/C)(C/A)AAGGX(C/T)X-3Ј (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). LRH-1 is closely related to the orphan receptor SF-1, which also binds DNA as a monomer and has important functions in the development and differentiation of steroidogenic tissues and in steroidogenesis and sexual determination (15). LRH-1 and SF-1 have more than 95% amino acid sequence identity in their DNA binding domains and recognize the same DNA sequences. These and other NR5A subfamily members contain a distinctive 26-amino acid motif, called the Fushi Tarazu-Factor1 domain, which is located just C-terminal to the conserved zinc finger DNA binding domain and contacts the upstream motif in the binding site (16).
Nuclear hormone receptors are typically ligand-dependent transcription factors. The ligand binding domain adopts a three-layer ␣-helical structure and upon ligand binding the C-terminal helix 12 is held in close proximity to the ligand binding pocket, forming a surface recognized by transcriptional coactivators (17,18). An initial crystal structure of the mouse LRH-1 ligand binding domain revealed that, unlike most other superfamily members, the LBD can form a stable active monomeric structure in the absence of ligand, coactivator peptide, or heterodimeric receptor partner, suggesting that LRH-1 may function as a constitutive activator (19). In contrast, several groups have recently shown by x-ray crystallography that the ligand binding pocket of bacterially expressed human LRH-1 is occupied by distinct phospholipids, including phosphatidlyethanolamine (20 -22), suggesting that human and mouse LRH-1 may be regulated quite differently. However, both mutagenesis results (20) and the ability of human and mouse SF-1 to bind similar phospholipids indicate that such potential agonists may target both mouse LRH-1 and other mammalian NR5A family members. Krylova et al. (21) suggested that phosphatidylinositols, major intracellular signaling molecules, bind to hLRH-1, raising the possibility of a direct linkage between phospholipid signaling and gene transcription. However, the functional roles of phosphoinositides and other potential phospholipid ligands as physiologic modulators of LRH-1 or SF-1 transactivation remain to be elucidated.
Several other physiological potential roles of LRH-1 have been suggested in recent studies. These include regulation of estrogen production by activation of aromatase gene expression (11) and activation of proliferation by induction of cyclin E and cyclin D1 expression (13). Unfortunately, the analysis of LRH-1 function using the knock-out approach, which has been so successful for other nuclear receptors, is precluded by the fact that homozygous loss of LRH-1 function results in very early embryonic lethality (13,31,32). This highlights the importance of alternative strategies to identify potential functional roles of this orphan and the pathways that may modulate them.
We have examined the potential impact of mitogenic stimuli on LRH-1 transactivation. We find that that LRH-1 hinge and ligand binding domains are sufficient to confer strong stimulatory effects of phorbol esters on LRH-1 transactivation in HeLa cells. ERK phosphorylates LRH-1 at at least two hinge domain serine residues, Ser-238 and Ser-243. Surprisingly, these phosphorylation sites do not correspond to the stimulatory site identified previously in SF-1 (33). Mutation of both LRH-1 residues to alanine largely eliminates PMA-dependent phosphorylation of LRH-1 in cells and decreases PMA-dependent transactivation, while mutations to positively charged aspartate residues that mimic phosphorylation increase basal activity. These results identify a novel response of LRH-1 to proliferative signals that could contribute to its reported stimulatory effects on cell cycle.

EXPERIMENTAL PROCEDURES
Materials and Plasmids-The reagents were obtained from the following sources: mitogen-activated protein kinase (MAPK) inhibitors, PD 98059, and SB 202190 were from CalBiochem; SP 600125 was a kind gift from Dr. Karin at University of California, San Diego; PMA, anti-FLAG antibody (M2), anti-FLAG M2-agarose were from Sigma; rhEGF and rhTNF␣ were from R&D systems; and U0126 was from Promega. CDM8His-Flag-hLRH-1 was created by PCR amplification of hLRH-1-(1-541) insert in pCI vector (from Dr. Belanger). All deletion and point mutation constructs were created using a PCR-based method. pCMX hLXR␣ and a luciferase reporter containing a 3.6-kb rat Cyp7A promoter (rat Cyp7Aluc) were kind gifts from Dr. J. Chiang of Northeastern Ohio Universities College of Medicine.
Transient Transfection-HeLa and HepG2 cells were used for transfection assays. Cells were transfected using the calcium phosphate-mediated method as described (34). Confluent cells were plated into 24-well plates with a 1:4 ratio 1 day before transfection. Approximately 16 h after transfection, cells were replenished with fresh DMEM and, if needed, treated with PMA or EGF. For treatment of MAPK inhibitors, these inhibitors were added 30 min before PMA or EGF treatment. Cells were harvested after 24-h incubation for luciferase assays. Actin-␤-galactosidase plasmids or TKGH were cotransfected for transfection normalization by quantitation of ␤-galactosidase or GH activities as described previously.
Metabolic Labeling-HeLa cells in 60-mm dishes were transfected with 8 g of CDM8flag-hLRH-1 or CDM8flag-mSHP plasmids using calcium phosphate method. After overnight incubation, media were replaced with fresh DMEM containing 1 or 10% FBS as indicated. 24 h later, cells were washed with phosphate-free DMEM and replenished with 1 ml of serum-free DMEM for cells maintained in 1% FBS or DMEM containing 1% dialyzed FBS for cells maintained in 10% FBS. After 1-h incubation, cells were labeled with 1 mCi of [ 32 P]orthophosphate for 4 h. PMA induction was performed during the last 1 h of the labeling. For HepG2 cells, cells were transfected with 10 g of CDM8flag-hLRH-1 plasmids and maintained with DMEM containing 10% FBS. During the labeling with 1 mCi of [ 32 P]orthophosphate for 1 h 20 min, one plate was treated with 20 M U0126 simultaneously to  (PD, 20 mM) and were further incubated for 24 h. TKGH was cotransfected as an internal transfection control. B, the strong, specific MEK1/2 inhibitor, U0126 (20 mM), was used as described for A to inhibit PMA and EGF effects. A ␤-actin promoter-driven ␤-galactosidase was used as transfection control, and luciferase values were normalized with ␤-galactosidase activity and plotted as means of triplicates.RLU, relative light units.
inhibit MAPK activity as indicated in Fig. 6B. PMA induction was performed during the last 20 min of the labeling. Then, cells were lysed in 500 l of RIPA buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 2 mM EDTA, 1 mM sodium vanadate, and proteinase inhibitors). The cell lysates were incubated with anti-FLAG-conjugated protein G-agarose (Sigma) overnight at 4°C, and the bound proteins were subjected to Western analysis using anti-FLAG antibodies. The same membrane was subjected to autoradiography to detect phosphorylated proteins.
In Vitro Phosphorylation-Bacterially expressed GST fusions were purified using glutathione-Sepharose 4B (Pharmacia Corp.). Beadbound GST fusions were incubated with 0.5 l of ERK2 (New England Biolabs) and 5 Ci of [␥-32 P]ATP (PerkinElmer Life Sciences) in 50 l of the manufacturer's buffer for 30 min at 30°C. Phosphorylated proteins were eluted with SDS sample buffer and resolved by SDS-PAGE. Gels were stained with Coomassie Blue, dried, and subjected to autoradiography.

Stimulation of LRH-1 Transcriptional Activity by PMA and EGF-
CYP7A1 encodes the first and rate-limiting step of the classical pathway in bile acid biosynthesis from cholesterol, and its expression is tightly controlled by several nuclear hormone receptors including LRH-1 (25)(26)(27). Nuclear receptors and AP-1 often show mutually antagonistic interactions, and a reported inhibitory effect of PMA on CYP7A1 promoter activity was mapped to a potential AP-1 site in the rat promoter (36) that overlaps with a potential LRH-1 binding site. Thus, the role of LRH-1 and other nuclear receptors in responses to PMA and other stimuli was examined in transient transfections of HeLa cells.
In agreement with the previous conclusion that LRH-1 functions in this context as a competence factor to promote responses to LXR and other factors (23), cotransfection with LRH-1 alone had no effect on CYP7A1 promoter activity (Fig. 1A). Also consistent with previous results, LXR␣ had a negative effect on promoter activity in the absence of oxysterols (37), and TNF␣ repressed promoter activity regardless of coexpressed LXR or LRH-1 (38,39). In contrast to earlier studies, however, PMA strongly stimulated CYP7A1 promoter activity in an LRH-1-dependent manner.
To examine the role of LRH-1 in this response, a reporter construct containing five SF-1/LRH-1 binding sites (CAAAGGTCA) from the basal 21-hydroxylase promoter (40), was used (34). Consistent with our earlier results, this construct was modestly activated by LRH-1 alone. Much more dramatically and in agreement with the CYP7A1 promoter results, it was strongly stimulated by PMA in the presence of LRH-1 (Fig. 1B). This effect was largely dependent on LRH-1 cotransfection, although PMA alone had a modest stimulatory effect in some experiments (data not shown). EGF alone had a more modest positive effect on LRH-1 transactivation. Transactivation directed by a fusion of the LRH-1 hinge and ligand binding domain (amino acids 185-541) to the heterologous Gal4 DNA binding domain (Gal4-LRH-1(L)) was also stimulated upon PMA treatment (Fig. 1C). Thus, LRH-1 residues C-terminal to 184 are sufficient to confer PMA responsiveness in the absence of CYP7A1 promoter-specific factors or the LRH-1 N terminus and DNA binding domain.
PMA is a well known protein kinase C activator that strongly stimulates the MAPK pathway (41,42), which includes ERK, JNK, and p38 kinases (43). Thus, several specific kinase inhibitors were used to examine the roles of these MAPKs in the PMA stimulation of LRH-1 activity. HeLa cells were transfected with the SF-1luc reporter and hLRH-1 expression vector and were treated with various MAPK inhibitors 30 min prior to PMA treatment. SP60012, a specific inhibitor for JNK, failed to block PMA stimulation, the p38-specific inhibitor SB202190  and was also exposed to x-ray film (bottom panel).

LRH-1 Phosphorylation
modestly decreased PMA stimulation, and the MEK1/2 inhibitor PD98059 had a stronger effect ( Fig. 2A). This was confirmed with the potent MEK1/2 inhibitor U0126, which completely blocked PMA-and EGF-mediated stimulation of LRH-1 activity (Fig. 2B). These results indicate that the ERK pathway mediates the stimulation of LRH-1 transcriptional activity by PMA or EGF in HeLa cells.
LRH-1 Is a Phosphoprotein in Vivo and in Vitro-Thus far, it is clear that transcriptional activity of LRH-1 is up-regulated by kinase signaling pathways, but it is unclear whether this is associated with direct LRH-1 phosphorylation. To determine whether mitogenic signals affect LRH-1 phosphorylation, HeLa cells expressing epitope-tagged LRH-1, or SHP for comparison, were in vivo labeled using [ 32 P]orthophosphate in the presence or absence of PMA. Immunoprecipitation and autoradiography revealed that both receptors are phosphoproteins and that LRH-1 phosphorylation was markedly increased by 1 h of PMA treatment (Fig. 3).
The ability of the specific MEK1/2 inhibitors PD98059 and U0126 to block the stimulation of LRH-1 activity by PMA is consistent with the fact that many previously identified nuclear receptor phosphorylation sites are targeted by MAPKs and also the more specific observation that ERK phosphorylation of serine 203 of SF-1, the closest relative of LRH-1, potently activates its transactivation (33). Thus, we tested the ability of purified ERK to phosphorylate LRH-1 in vitro.
Several fragments of LRH-1 fused to GST depicted in Fig. 4A were expressed in Escherichia coli and purified using glutathione-Sepharose beads. The beads were incubated with purified ERK2 in the presence of [␥-32 P]ATP, and bound proteins were eluted, resolved by SDS-PAGE, and visualized by Coomassie Blue staining or autoradiography. As expected, the full-length LRH-1 GST fusion (F) was phosphorylated by ERK (Fig. 4B, lane 5). Among the fragments tested, only ⌬2 and the hinge plus ligand binding domain (L) were efficiently labeled (Fig. 4B,  lanes 3 and 6), indicating that ERK phosphorylation sites reside between residues 185 and 386.
Role of Serines 238 and 243 in ERK Phosphorylation and PMA Stimulation-Inspection of this region identified four proline-directed serine residues, i.e. potential MAPK phosphorylation sites, at positions 238, 243, 280, and 297. Since fragment ⌬2 contains only three of these four residues, mutations were introduced into the slightly larger GST-LRH-1 ⌬3 fragment. Individual S238A and S243A mutants showed reduced ERK phosphorylation, and mutation of both residues completely abolished in vitro labeling (Fig. 5A, upper panels). This result was somewhat surprising, since Ser-280 corresponds much more closely to the SF-1 ERK target Ser-203 (33) (Fig. 7). The role of the two more proximal sites was more critically tested by introducing the same mutations individually or together into the full-length GST-LRH-1. As observed with the ⌬3 fragment, each single mutation strongly decreased phosphorylation, and it was abolished by the double mutation (Fig. 5A,  lower panels).
These results raise the question of the role of these residues in the PMA-dependent in vivo phosphorylation of LRH-1. By comparison to the PMA stimulated labeling of full-length wild type LRH-1 in HeLa cells, labeling of the S238A,S243A double mutant was markedly reduced (Fig. 5B). Thus, these sites are required for the majority of the PMA-dependent phosphorylation of LRH-1 in these cells.
These results clearly predict that the S238A and S243A mutations should inhibit PMA stimulation of LRH-1 transactivation, while complementary phospho-mimic mutations to negatively charged aspartate residues should increase basal transactivation. Thus, wild type LRH-1 and S238A,S243A and S238D,S243D double mutants were transfected into HeLa cells. To minimize any effect of growth factors present in FIGURE 5. Ser-238 and Ser-243 are required for PMA dependent LRH-1 phosphorylation. A, the indicated Ser to Ala mutations were introduced into ⌬3 and the full-length GST-LRH-1 and subjected to in vitro phosphorylation as described in the legend to Fig. 4. B, HeLa cells were transfected with 8 g of FLAG-tagged LRH-1 wild type or S238A,S243A mutant plasmid as indicated using the CaPO 4 -medaited method. Next morning, cells were washed with PBS and replenished with serum-free DMEM. Cells were processed as described in the legend to Fig. 3 except that cells were radiolabeled for 3 h in phosphate-free DMEM containing 1% FBS. Arrows point to specific LRH-1 bands. C, HeLa cells were transfected with SF-1luc reporter along with LRH-1 wild type or S238A,S243A mutant plasmids. The cells were treated with 50 ng/ml PMA or 200 ng/ml EGF for 24 h in serum-free DMEM. Then the cells were harvested for luciferase assay. D, HeLa cells were transfected with SF-1luc along with wild type LRH-1 or S238D,S243D mutant plasmid. 12 h after transfection, the cells were replenished with fresh serum-free DMEM and further incubated for 24 h. E, the indicated Gal4, Gal4LRH-1(L) wild type (WT), or mutant plasmids (S/A, S238A,S243A; S/D, S238D,S243D) were transfected into HeLa cells along with Gal4TKluc reporter gene constructs. Their transcriptional activities were measured as luciferase reporter activities at 40 h after transfection. All the transfection assays were normalized by cotransfected ␤-galactosidase activities and presented as means of triplicates. MARCH 24, 2006 • VOLUME 281 • NUMBER 12 serum, transfected cells were maintained in serum-free medium with or without PMA treatment. As expected, the PMA response of the S238A,S243A double mutant was significantly decreased relative to wild type LRH-1, while the basal activity of the S238D,S243D double mutant was increased (Fig. 5, C and D). The introduction of an Ser to Ala mutation at residue 280 of LRH-1 failed to change any transcriptional activity (data not shown). In the context of the Gal4-LRH-1 hinge and ligand binding domain fusion, the Ser to Ala double mutant had a more dramatic negative effect on transactivation, while the effect of the Ser to Asp double mutant was more modest (Fig. 5E).

LRH-1 Phosphorylation
Serines 238 and 243 Are Functional in ERK Phosphorylation and PMA Stimulation in a Human Liver Cell Line, HepG2-Since liver is a primary site for LRH-1 function, we tested the PMA inducibility of LRH-1 transcriptional activity in the human hepatoma cell line HepG2. As in HeLa cells, LRH-1 activity was increased by PMA treatment, and this response was not affected by the JNK inhibitor SP60012 but was greatly hampered by pretreatment with the p38 inhibitor SB202190 and the MEK1/2 inhibitor PD98059 (Fig. 6A). To test the phosphorylation of the Ser-238 and Ser-243 residues in HepG2 cells, metabolic labeling was performed with FLAG-tagged wild type and S238A,S243A double mutant LRH-1 plasmids. As observed in HeLa cells, LRH-1 phosphorylation was strongly increased in response to PMA treatment, and this was blocked by both pretreatment with U0126 and the double S238A,S243A mutation (Fig. 6B). These results suggest that the modulation of LRH-1 transactivation by PMA is regulated through similar pathways in the HeLa and HepG2 cells.

DISCUSSION
Overall, these results demonstrate that the Ser-238 and Ser-243 residues of the LRH-1 hinge are required for effective in vitro phosphorylation by ERK and also for PMA-dependent in vivo phosphorylation. Preventing Ser-238 and Ser-243 phosphorylation blunts but does not eliminate the stimulatory effect of PMA on LRH-1 transactivation, while the S238D,S243D double phosphomimic mutant shows an ϳ2 fold increase in transactivation that is comparable to phosphorylation dependent effects on other receptors. For example, ER␣ transactivation is increased 1.8 fold by overexpression of Ras in COS cells (44), and the S305E mutant of ER␣ exhibits only 30% higher transactivation in a human breast cancer cell line (45). We conclude that PMA dependent direct phosphorylation at these two positions stimulates LRH-1 transactivation in, at least, HeLa and HepG2 cells.
The functional role of this hinge region is strongly supported by a recent report that the segment between 186 and 219 in a human LRH-1 isoform, which corresponds to 236 -265 of in the full-length human LRH-1 numbering used here, contains an additional activation function of LRH-1 (46). However, the contrast between the residual PMA responsiveness of the S238A,S243A double mutant and the complete blockade by U0126 indicates that additional targets of the ERK pathway contribute to the increased transactivation. The relatively modest residual in vivo phosphorylation of the S238A,S243A double mutant (Fig. 5B) suggests that additional LRH-1 sites could be involved in HeLa cells. However, recent results emphasize the potential role of phosphorylation of other targets, particularly the p160 coactivators that can be activated by ERK and other pathways (47)(48)(49)(50)(51). Thus, it seems likely that cofactor activation could also contribute to the stimulation of LRH-1 transcriptional activity.

FIGURE 6. Stimulation of LRH-1 transactivation by PMA is conserved in HepG2 cells.
A, HepG2 cell were transfected, and some cells were treated with the indicated MAPK inhibitors before PMA stimulation as described in the legend to Fig. 2A. The luciferase values are means of triplicates after normalization by cotransfected ␤-galactosidase activities. B, HepG2 cells were subjected to metabolic labeling after being transfected with 10 g of the indicated FLAG-tagged hLRH-1 plasmids using the CaPO 4 -mediated method to verify in vivo phosphorylation. Total labeling with 1 mCi of orthophosphate lasted 1 h 20 min. 20 M U0126 treatment also lasted 1 h 20 min, and PMA induction was performed during the final 20 min. Other procedures were identical to the one described in the legend to Fig. 3 except that a whole 500 ml of cell extracts were subjected to immunoprecipitation. The arrows point to the specific LRH-1 bands.

LRH-1 Phosphorylation
The results with LRH-1 are also generally consistent with the characterization of a similar phosphorylation-dependent activation of its close relative, SF-1. Surprisingly, however, the specific phosphorylation targets differ in the two receptors. The human LRH-1 sites identified here are not found in SF-1 (Fig. 7A) but are conserved in rodent LRH-1 sequences (Fig. 7B). In contrast, the region surrounding LRH-1 Ser-280 is conserved in SF-1, but this LRH-1 residue does not align the phosphorylated serine in SF-1 but with the adjacent proline (Fig. 7B). The discrepancy in hinge phosphorylation targets in the two receptors is not due to different cell contexts, since both were studied in HeLa cells, which are commonly used to characterize such signaling responses. However, it remains possible that additional pathways targeting LRH-1 Ser-280 or other potential sites will be identified in other contexts.
The current results identify a PMA-dependent pathway for stimulation of LRH-1 transactivation and raise the important question of the impact of this pathway on normal LRH-1 function, including its role in the intriguing potential responses to phospholipid or other ligands. Since ERK activation is generally a proliferative stimulus, it is an attractive possibility that phosphorylation of Ser-238 and Ser-243 may support the proposed role of LRH-1 as a proliferative factor (13). It will be necessary to develop appropriate phospho-specific antisera to test the prediction that mitogenic stimuli increase Ser-238 and Ser-243 phosphorylation in vivo. In contrast, activation of stress-activated protein kinase pathways is associated with inhibitory effects on the hepatic LRH-1 target CYP7A1 (52). Interestingly, a recent study indicates that the p38 MAPK pathway is involved in LRH-1 protein expression in rat ovarian granulosa cells (3). It will be interesting to test whether p38 or other stress-activated kinases directly target the LRH-1 protein for phosphorylation or regulates LRH-1 expression or activity at other levels. As with other nuclear receptors, the impact of diverse signaling pathways on LRH-1 function is likely to be complex. The results described here provide both a firm rationale for such studies of LRH-1 and a good starting point for further exploration.