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Molecular Endocrinology, doi:10.1210/me.2008-0092
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Molecular Endocrinology 22 (11): 2433-2447
Copyright © 2008 by The Endocrine Society

Phosphorylation of Farnesoid X Receptor by Protein Kinase C Promotes Its Transcriptional Activity

Romain Gineste, Audrey Sirvent, Réjane Paumelle, Stéphane Helleboid, Alexis Aquilina, Raphaël Darteil, Dean W. Hum, Jean-Charles Fruchart and Bart Staels

GENFIT (R.G., A.S., S.H., A.A., R.D., D.W.H.), 59120 Loos-Lez-Lille, France; Institut Pasteur de Lille (R.P., J.-C.F., B.S.); Département d’Athérosclérose; Institut National de la Santé et de la Recherche Médicale Unité 545 (R.G., R.P., J.-C.F, B.S.); Lille F-59019, France; and Université de Lille 2 (R.P., J.-C.F., B.S.), Faculté de Pharmacie, Faculté de Médecine, Lille F-59006, France

Address all correspondence and requests for reprints to Bart Staels: Unité de Recherche 545 Institut National de la Santé et de la Recherche Médicale, 1 rue du Prof Calmette, Boîte Postale 245 59019 Lille, Cedex, France. E-mail: Bart.Staels{at}pasteur-lille.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKC{alpha} phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKC{alpha}-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor {gamma} coactivator 1{alpha}. In conclusion, these findings show that the phosphorylation of FXR induced by PKC{alpha} directly modulates the ability of agonists to activate FXR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE FARNESOID X RECEPTOR {alpha} (FXR, NR1H4) belongs to the nuclear receptor superfamily. The FXR{alpha} gene encodes four FXR isoforms (FXR{alpha}1, FXR{alpha}2, FXR{alpha}3, and FXR{alpha}4) in humans and mice (1, 2). FXR binds natural or synthetic ligands, such as bile acids or GW4064, respectively (3, 4, 5, 6). FXR expression is highest in the liver, intestine, kidney, and adrenal gland (2). A physiological role of FXR is to regulate bile acid homeostasis. In the liver, FXR prevents bile acid accumulation by decreasing both their biosynthesis and uptake, and promoting their excretion in bile. Indeed, FXR decreases expression of CYP7A1, the rate-limiting enzyme of the neutral bile acid biosynthesis pathway, by up-regulating the small heterodimer partner (SHP) in the liver and FGF15 in the intestine (7, 8). FXR promotes bile acid export by increasing bile salt export pump expression and prevents bile acid uptake by decreasing Na+-taurocholate cotransporting polypeptide expression (9, 10, 11). FXR also promotes conjugation of bile acids into tauro-, glyco-, glucuro- or sulfo-derivates by increasing the expression of enzymes such as bile acid-coenzyme A: amino acid N-acetyltransferase, bile acid-coenzyme A synthetase, UDP-glucuronosyltransferase 2B4 (UGT2B4), and steroid sulfotransferase A1 resulting in diminished bile acid hydrophobicity and toxicity (12, 13, 14). Moreover, FXR is implicated in high-density lipoprotein (HDL) and triglyceride (TG) metabolism. FXR-deficient mice display elevated HDL cholesterol levels associated with increased plasma apolipoprotein (apo) A-I concentrations (10). Furthermore, activated FXR represses apoA-I gene expression and induces expression of phospholipid transfer protein, an enzyme involved in HDL remodeling (15, 16). In addition, FXR–/– mice also display elevated serum TG concentrations (10). FXR promotes TG clearance by regulating the expression of the apoCII and apoCIII genes (17, 18) and also inhibits the hepatic production of TG by repressing the expression of lipogenic target genes (19, 20). Finally, more recently, several reports demonstrated a role of FXR also in the regulation of carbohydrate metabolism and insulin sensitivity (21, 22, 23, 24).

FXR modulates the expression of its target genes by binding either as a monomer or as a heterodimer with the retinoid X receptor (RXR, NR2B1) to DNA sequence motifs called FXR response elements (FXREs). Most FXR target genes, like SHP, are regulated by FXR/RXR heterodimers, which bind to an inverted repeat composed of two AGGTCA half-sites spaced by one nucleotide (IR-1) (7). However, it has been demonstrated that FXR can also bind as a monomer to half-site FXREs in the promoters of the UGT2B4 (induced) and apoA-I (repressed) genes (13, 15). As other nuclear receptors, the transcriptional activation of FXR involves the binding and recruitment of coactivators to and release of corepressors from target gene promoters (25). Indeed, binding of bile acids to FXR triggers the recruitment of steroid receptor coactivator 1 (SRC1), which belongs to the SRC/p160 family (3, 4, 26). Other FXR coactivators are the vitamin D receptor-interacting protein (DRIP205), PPAR{gamma} coactivator 1{alpha} (PGC-1{alpha}), and protein arginine methyl-transferases (PRMT) such as PRMT1 and PRMT4/coactivator associated arginine methyltransferase 1 (20, 27, 28, 29, 30). Finally, GPS2, a subunit of a conserved corepressor complex, was identified as a regulator of bile acid biosynthesis capable of interacting with FXR (31).

The activity of nuclear receptors can also be regulated by posttranslational modifications such as phosphorylation (32). However, so far, evidence of cross talk between kinase-signaling pathways and FXR activity has not been demonstrated. Interestingly, bile acids are able to activate kinase pathways. For instance, certain bile acids modulate protein kinase A (PKA) by binding to the G-protein-coupled receptor TGR5 and inducing cAMP production (33, 34, 35). Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is also triggered in a rat hepatoma cell line by bile acids such as taurochenodeoxycholic acid (36). Lastly, several studies have shown that bile acids such as tauroursodeoxycholic acid, chenodeoxycholic acid, and taurolithocholic acid activate protein kinase C (PKC) in hepatocytes (37, 38, 39, 40). Taurocholate represses the transcriptional activity of Cyp7AI by PKC{alpha} activation in primary cultures of rat hepatocytes (41). Deoxycholic acid induces Ca2+ accumulation in BHK-21 fibroblasts and, consequently, promotes activation of conventional PKC isoforms (42). Finally, ursodeoxycholic acid, used for the treatment of cholestatic liver diseases, stimulates hepatobiliary excretion of bile acids through PKC{alpha} activation (43).

The PKC family consists of serine/threonine-specific protein kinases (44). The PKC family is classified into three groups: 1) the conventional PKCs {alpha}, βI, βII, and {gamma} require, for their activity, negatively charged phospholipids, diacylglycerol (DAG) or phorbol ester and calcium; 2) the novel PKCs {delta}, {epsilon}, {theta}, {eta}, and µ require negatively charged phospholipids, diacylglycerol or phorbol ester, but not calcium; and 3) the atypical PKCs {lambda} and {xi} do not require calcium, diacylglycerol, or phorbol esters but only negatively charged phospholipids. They differ in primary structure, tissue distribution, subcellular localization, in vitro mode of action, response to extracellular signals, and substrate specificity. The activity of these PKCs can be modulated by several compounds. Phorbol 12-myristate 13-acetate (PMA) and Rö 31-8220 are, respectively, non-isoform-selective activators and inhibitors of PKC whereas Gö-6976 is a Ca2+-dependent PKC-selective inhibitor (45).

Our results show that inhibition of the Ca2+-dependent PKC results in a decreased transcriptional activity of FXR. Moreover, this study provides, for the first time, evidence that FXR is a substrate of PKC{alpha} and that the phosphorylation in its DNA-binding domain (DBD) is essential for a full ligand-dependent transactivation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PKC Inhibitors Inhibit Ligand Regulation of FXR Target Genes
Because bile acids modulate kinase pathways such as protein kinase C (37), protein kinase A (46), and PI3K/Akt (36), the effect of different protein kinase inhibitors was tested on the expression of FXR target genes such as SHP, UGT2B4, and apoA-I. HepG2 cells were pretreated for 2 h with Rö 31-8220, PKA inhibitor (PKI), or wortmannin, respectively, PKC, PKA and PI3K inhibitors, and subsequently treated with the natural FXR agonist CDCA. As expected (7, 13, 15), CDCA increased significantly SHP (Fig. 1AGo) and UGT2B4 (Fig. 1BGo) mRNA levels and decreased apoA-I (Fig. 1CGo) mRNA levels. Surprisingly, pretreatment with Rö 31-8220, but not PKI or wortmannin, inhibited CDCA-induced SHP and UG2B4 gene expression and reduced the down-regulation of apoA-I gene expression by CDCA.


Figure 1
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  		Fig. 1. PKC but not PKA or PI3K Inhibitors Inhibit the Regulation of FXR Target Genes by CDCA

HepG2 cells were treated with vehicle (white bars) or CDCA (75 µM) (black bars) for 24 h, in the absence (ctrl) or presence of the PKC inhibitor (Ro-31-8220 1 µM) (Ro), the PKA inhibitor (1 µM) (PKAI), the PI3K inhibitor (wortmannin, 250 nM) (wort). A, –SHP (panel A), – UGT2B4 (panel B), and – apo-AI (panel C) mRNA levels were measured by real-time quantitative PCR. Values are means ± SD (n = 3). Statistically significant differences between control and treated cells are indicated by asterisks [Student’s t test: ***, P < 0.001; **, P < 0.001; P < 0.01; *, P < 0.05; NS, not significant, P > 0.05]. Ctrl, Control; DMSO, dimethysulfoxide.

 
Because Rö 31-8220 is an inhibitor of all PKC isoenzymes, it was tested whether the calcium-dependent PKC signaling pathway is involved in the regulation of FXR target genes using the specific inhibitor Gö-6976. As expected, both CDCA and GW4064 regulated the three target genes. Preincubation with Gö-6976 decreased SHP and UGT2B4 mRNA expression induced by GW4064 and CDCA (Fig. 2Go, A and B) and prevented the repression of apo-AI gene expression (Fig. 2CGo).


Figure 2
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  		Fig. 2. A Calcium-Dependent PKC-Specific Inhibitor Inhibits the Regulation of FXR Target Genes by FXR Agonists

HepG2 cells were treated with vehicle (white bars), CDCA (75 µM) (black bars), or GW4064 (1 µM) (gray bars) for 24 h, in the absence (ctrl) or presence of the calcium-dependent PKC inhibitor Gö6976 (1 µM). SHP (panel A), UGT2B4 (panel B), and apo-AI (panel C) mRNA levels were measured by real-time quantitative PCR. Values are means ± SD (n = 3). Statistically significant differences between control and treated cells are indicated by asterisks [Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant, P > 0.05]. Ctrl, Control; DMSO, dimethylsulfoxide.

 
These data suggest that the calcium-dependent PKC signaling pathway modulates the regulation of FXR target gene expression by its ligands CDCA and GW4064.

Calcium-Dependent PKC Promotes FXR Transcriptional Activity
To determine whether PKC modulates the transcriptional activity of FXR, HepG2 cells were transfected with a reporter vector containing three copies of the FXRE of the human ileal bile acids-binding protein (I-BABP) gene promoter (TkpGL3-3xFXRE-pLuc) and the pCDNA3-hFXR expression plasmid. The transfected cells were pretreated with or without the PKC activator PMA or inhibitors Rö 31-8220 and Gö-6976 before incubation with the FXR agonist GW4064. In the absence of PKC inhibitors, GW4064 induced FXR transcriptional activity significantly. Pretreatment with Rö 31-8220 and Gö-6976 significantly decreased ligand-dependent FXR transactivation, whereas PMA strongly enhanced FXR activity to 80-fold (Fig. 3Go). Rö 31-8220, Gö-6976, and PMA did not significantly modulate protein levels (data not shown). Thus, PKC modulates ligand-dependent FXR transactivation. Furthermore, the effect of Gö-6976 suggests that calcium-dependent isoforms of PKC modulate FXR transactivation.


Figure 3
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  		Fig. 3. The PKC Signaling Pathway Controls the Transcriptional Activity of FXR

HepG2 cells were cotransfected during 12 h with luciferase control plasmid TkpGL3 (100 ng) as negative control, or luciferase reporter plasmid TkpGL3–3xFXRE (100 ng) driven by three copies of the positive FXRE of the human IBABP promoter in the presence of pcDNA3-hFXR (20 ng) and pSG5-RXR{alpha} (20 ng). Cells were treated during 6 h with vehicle (white bars) or 500 nM of GW4064 (gray bars) in the absence (ctrl) or presence of PKC modulators: Rö-31–8220 (Rö, 1 µM), Gö6976 (Gö, 1 µM), or PMA (100 nM). Results are expressed in fold induction and normalized to internal control Renilla activity. Values are means ± SD (n = 3). Statistically significant differences between control and treated cells are indicated by asterisks [Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant, P > 0.05]. Ctrl, Control; DMSO, dimethylsulfoxide.

 
Endogenous FXR Is a Phosphoprotein Phosphorylated by PKC in HepG2 Cells
Next, we addressed whether FXR is a phosphoprotein and whether PKC modulates its phosphorylation state. Proteins extracted from HepG2 cells were treated or not with calf intestinal phosphatase (CIP) before passage through a phosphoprotein affinity column (47). The presence of endogenous FXR in lysate and elution fraction was analyzed by Western blotting using a FXR antibody. FXR was eluted in the phosphoprotein fraction whereas pretreatment with CIP phosphatase completely abolished its presence in this fraction (Fig 4AGo). This demonstrates that endogenous FXR is phosphorylated in HepG2 cells. To study whether PKC is involved in the phosphorylation of endogenous FXR, a two-dimensional gel analysis-based approach was used. HepG2 cells were pretreated or not with the PKC inhibitor Rö 31-8220 for one 1 h and subsequently treated or not with PMA (15 min). Proteins were extracted and separated on a two-dimensional SDS-PAGE gel, blotted on a nitrocellulose membrane, and subjected to immunodetection using a FXR antibody (Fig. 4BGo). FXR immunodetection revealed two spots in untreated HepG2 extracts likely representing nonphosphorylated endogenous FXR protein, and at more acidic pH, a phosphorylated FXR form (Fig. 4BGo, upper panel). After PMA treatment, supplemental spots with a more acidic pH isoelectric appeared (Fig. 4BGo, middle panel), whereas pretreatment with the PKC inhibitor Rö 31-8220 prevented this PMA-induced pattern change (Fig. 4BGo, lower panel). These data suggest that the spots appearing upon PMA treatment correspond to different PKC-phosphorylated FXR isoforms.


Figure 4
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  		Fig. 4. FXR is a Phosphoprotein and Its Phosphorylation Is Induced by the PKC Activator PMA

A, Proteins extracted from HepG2 cells were treated or not with CIP and purified on phosphoprotein affinity columns. B, Proteins were extracted from HepG2 cells pretreated or not (Ctr) with Rö-31-8220 (Rö), subsequently treated or not (Ctr) with PMA (100 nM) for 15 min, and separated by two-dimensional gel analysis, blotted to a nitrocellulose membrane and subjected to immunodetection using a FXR antibody. Ctr, Control; pHi, pH isoelectric.

 
FXR Is Phosphorylated in its DNA-Binding Domain on Residues S135 and S154
To identify the PKC phosphorylation sites in FXR, different recombinant fragments covering the full-length FXR protein were produced, purified, and subjected to an in vitro phosphorylation assay (Fig. 5AGo). Because calcium-dependent PKCs modulate FXR activation, these fragments were incubated in the presence of recombinant PKC{alpha}, βI, βII, or {gamma}. Phosphorylation was detected by Western blot analysis using phospho-specific antibodies. Myelin basic protein, used as a positive control, was in vitro phosphorylated by all calcium-dependent PKC (Fig. 5BGo, upper panel). The use of a phosphothreonine antibody did not result in the detection of FXR phosphorylation (data not shown). However, the phosphoserine antibody revealed that the FXR [106–196 amino acids (AA)] protein fragment, containing the DNA-binding domain, was preferentially phosphorylated by PKC{alpha} and βI (Fig. 5BGo, lower panel).


Figure 5
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  		Fig. 5. The DBD of FXR Is in Vitro Phosphorylated by PKC{alpha} and PKCβI

A, Schematic diagram of GST-fused fragments covering the entire length of FXR (left panel). FXR 1-105AA (lane 1), FXR 106-196AA (lane 2), FXR 197-284AA (lane 3), and FXR 215-472 AA (lane 4) fragments were purified, and GST was removed by factor Xa cleavage. Proteins were separated by SDS-PAGE and revealed with Coomassie staining (right panel). The arrows indicate the purified FXR fragments. B, Myelin basic protein (upper panel) and FXR 1-105 AA (lane 1), FXR 106-196 AA (lane 2), FXR 197-284 AA (lane 3) and FXR 215-472 AA (lane 4) fragments (lower panel) were incubated (+) or (–) not with recombinant calcium-dependent PKC isoforms and subjected to Western blot analysis using antiphosphoserine. LBD, Ligand-binding domain; MBP, myelin basic protein.

 
This fragment contains three serines, S117, S135, and S154, which were mutated to alanine (Fig. 6AGo). Wild-type and mutated fragments were purified and subjected to an in vitro phosphorylation assay using PKC{alpha}. The replacement of S135 and S154 by alanine significantly decreased the phosphorylation of the FXR DBD fragment (Fig. 6BGo).


Figure 6
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  		Fig. 6. Mutagenesis of S135 and S154 Prevents the Phosphorylation of FXR DBD by PKC{alpha}

A, Schematic diagram of the FXR (106-196 AA) fragment sequence showing the localization of serines (bold). Sequences corresponding to zinc fingers domains are underlined (left panel). S117, S135, and S154 were mutated into alanine in the GST-FXR (106-196 AA) fragment. Fragments were purified, analyzed by SDS PAGE, and stained with Coomassie blue (right panel). The arrow indicates the purified FXR fragments. B, FXR fragments, wild types, and mutants, were incubated (+) or (–) not with recombinant calcium-dependent PKC{alpha} and subjected to Western blot analysis using antiphosphoserine antibody. C, –HEK293 cells were transfected with FXR wild type-pCDNA3 and FXR S135AS154A-pCDNA3 and treated with PMA (100 nM) for 15 min. Proteins extracts were treated (+) or (–) not with CIP and purified on phosphoprotein affinity columns. LBD, Ligand-binding domain; WT, wild type.

 
To confirm these data in a cellular context, human embryonic kidney (HEK)293 cells were transfected with wild-type or mutant (S135AS154A) FXR and treated with PMA to activate PKC. Proteins were extracted, treated or not with CIP, and purified on the phosphoprotein affinity columns after which the presence of FXR protein was revealed in the different fractions by Western blot analysis using an FXR antibody. Analysis of lysates show that the expression level of FXR wild-type and FXR mutant proteins was similar (Fig. 6CGo). FXR was eluted in the phosphoprotein-enriched fraction, whereas CIP treatment abolished this elution pattern, showing that FXR is also phosphorylated in PMA-stimulated HEK293 cells (Fig. 6CGo). The replacement of S135 and S154 by alanine decreased the amount of phospho-FXR in the elution fraction, demonstrating that, in cells, FXR is phosphorylated by PMA-stimulated PKC via the S135 and S154 sites.

The FXRS135AS154A Mutant Displays Impaired PKC{alpha}-Induced FXR Activation
Because FXR is phosphorylated in vitro by PKC{alpha} on S135 and S154, and because calcium-dependent PKCs promote FXR activity, we next tested the functional consequences of mutations of these two amino acids on PKC-induced FXR activity. Cells were cotransfected with the wild-type or mutated FXR and the TkpGL3-3xFXRE-pLuc reporter construct in the presence of a constitutive active PKC{alpha} (PKC{alpha}CA). As with the PKC activator PMA, PKC{alpha}CA strongly increased the CDCA-induced FXR activity (Fig. 7AGo). Both mutants FXR S135A and FXR S154A exhibited a partially impaired transactivation by PKC{alpha}CA. Moreover, the double mutation completely prevented the enhancement of CDCA induction by PKC{alpha}CA. The wild-type and mutant FXR proteins were equally expressed in HEK293 cells, and PKC{alpha}CA did not modify FXR expression levels (Fig. 7BGo). These data indicate that the S135 and S154 sites in FXR mediate its transcriptional activation by PKC{alpha}.


Figure 7
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  		Fig. 7. PKC{alpha} Regulates FXR Activity via S135 and S154

A, HEK293 cells were cotransfected for 12 h with TkpGL3-3xFXRE-pLuc (100 ng), pcDNA3-FXR (1 ng) (wild type or mutants) and pSV40 (1 ng) as negative control or 1 ng PKC{alpha}CA. Cells were treated during 12 h with vehicle (white bars) or CDCA (black bars). Results are expressed in fold induction. Values are means ± SD (n = 3). Statistically significant differences between control and treated cells are indicated by asterisks (Student’s t test: ***, P < 0.001; **, 0.001 < P < 0.01; *, 0.01 < P < 0.05; NS, not significant). B, Wild type, FXR mutants, and PKC{alpha}CA were expressed in HEK293, and proteins were analyzed by immunoblotting (IB) using FXR and PKC{alpha} antibodies. Equivalent loading is shown by the actin signal. DMSO, Dimethylsulfoxide; WT, wild type.

 
The FXRS135AS154A Mutant Exhibits an Impaired Ligand-Dependent Activation
Because simultaneous mutation of S135 and S154 totally abrogated the up-regulation of FXR transactivation by PKC{alpha}, the response to agonists was compared between the wild-type and FXRS135AS154A mutant in a transactivation assay. HepG2 cells were transfected with the TkpGL3-3xFXRE-pLuc reporter and pCDNA3-hFXR or pCDNA3-hFXR S135AS154A and subsequently treated with increasing concentrations of synthetic or natural agonists. The fold inductions by GW4064 (Fig. 8AGo) and CDCA (Fig. 8BGo) were significantly lower for FXRS135AS154 than for wild-type FXR. The fold induction of FXR S135AS154A mutant transactivation by CDCA was decreased by 80%, whereas the fold induction of GW4064 was decreased by 30%. Western blot analysis using a FXR antibody showed that wild-type and mutant FXR proteins were overexpressed to equal levels in HepG2 cells (Fig. 8CGo). These data demonstrate that double mutation of the PKC phosphorylation sites impairs ligand activation of FXR.


Figure 8
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  		Fig. 8. The FXRS135AS154A Mutant Is Less Responsive to GW4064 and CDCA

HepG2 cells were cotransfected for 12 h with TkpGL3-3xFXRE-pLuc (100 ng) and pcDNA3-FXR (1 ng) (wild type or mutant). Cells were treated during 12 h with increasing concentrations of GW4064 (A) and CDCA (B). Results are expressed in fold induction. C, Wild-type and FXR mutant were expressed in HepG2 cells, and proteins were analyzed by immunoblotting (IB) using FXR antibody. Equivalent loading is shown by the actin signal. WT, wild type.

 
Phosphorylation of FXR by PKC Increases Neither Its DNA Binding Affinity nor Its Subcellular Localization
Because the FXR residues phosphorylated by PKC{alpha} are localized within the DBD, we studied whether they may influence the binding of FXRS135AS154A to a FXRE by EMSA. The FXR S135AS154A mutant did not exhibit a major difference of binding on the FXRE as compared with wild-type FXR (Fig. 9AGo). Moreover, in vitro FXR phosphorylation with PKC{alpha} did not increase the affinity of FXR for its FXRE. To determine whether PKC activation of FXR could occur in a FXRE-independent manner, HepG2 cells were transfected with an expression vector coding a chimeric protein consisting of the DNA-binding domain of the Saccharomyces cerevisiae GAL4 transcription factor fused to the full-length FXR (GAL4-FXR FL) and a reporter vector driven by five response elements for the GAL4 protein and treated with GW4064 in the presence of PMA. Interestingly, PMA treatment enhanced GW4064-induced transactivation of the GAL4-FXR full-length wild-type protein (Fig. 9BGo), whereas GAL4-FXRS135AS154A transactivation was not modified. Altogether, these data indicate that PKC-activation of FXR transcription does not occur via modulation of FXR DNA-binding capacity.


Figure 9
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  		Fig. 9. Phosphorylation of FXR Does Not Influence Its FXRE Binding Capacity

A, EMSA was performed using a [{gamma}-32P]ATP-labeled oligonucleotide probe containing the wild-type I-BABP IR-1 FXR [FXRE (IR1)] or mutated (FXREmut) binding site in the presence of reticulocytes lysate expressing or not FXR (wild type or S135AS154A) and RXR. Reticulocytes were incubated (+) or not (–) with PKC{alpha} (right panel). Arrow indicates the DNA-bound heterodimer FXR/RXR. B, HepG2 cells were cotransfected during 12 h with a luciferase expression plasmid (UAS)x5-TK-Luc (100 ng) in the presence of pcDNA3-GAL4 (5 ng), as negative control, or pCDNA3-GAL4-FXR full-length (FL) (wild type or mutant). Cells were treated during 6 h with vehicle (white bars) or GW4064 (1 µM; gray bars) in the absence (ctr) or presence of PMA (100 nM). The results are expressed as fold induction and normalized to internal control Renilla activity. ctr, Control; DMSO, dimethylsulfoxide; WT, wild type.

 
To investigate whether the effect of PKC activation could influence subcellular FXR localization, plasmids coding for a green fluorescent protein (GFP)-FXR fusion protein were transfected in HEK293 cells that were treated or not with PMA (supplemental Fig. 1 published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org). GFP-FXR was essentially localized in the nucleus, and PMA did not modify its subcellular localization. Time-course experiments with PMA did not modify FXR localization (data not shown).

FXR Phosphorylation by PKC Promotes the Recruitment of the Coactivator PGC1{alpha}
The coactivator PGC1{alpha} has been described to interact with the FXR DBD (20). Therefore, we analyzed whether PKC-induced FXR phosphorylation could influence its interaction with PGC1{alpha}. Glutathione-S-transferase (GST) pull-down assays confirmed that PGC1{alpha} (AA 1-400) indeed physically interacts with the FXR DBD (Fig 10AGo). Interestingly, in vitro phosphorylation of the FXR DBD by PKC{alpha} increased the recruitment of PGC1{alpha} (AA 1-400) (Fig. 10AGo). Western blot analysis using a GST antibody confirmed that equal amounts of GST proteins were incubated with PGC1{alpha} (1-400) and, using a phosphoserine antibody, that the FXR DBD was phosphorylated by PKC{alpha}.


Figure 10
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  		Fig. 10. Phosphorylation of FXR Promotes the Recruitment of the Coactivator PGC1{alpha}

A, Purified GST or GST-FXR DBD proteins were phosphorylated (+) or not (–) with PKC{alpha}. GST pull-down assays were performed using 20 µg of GST-fused FXR ligand-binding domain protein and 5 µg of His-PGC1{alpha}. Proteins were analyzed by Western blotting using GST, phosphoserine, and His antibodies (left panel). Bands corresponding to His-PGC1{alpha} were quantified by densitometry. Values expressed as percentage were plotted (right panel). Statistically significant difference is indicated by asterisk (Student’s t test: ***, P < 0.001; **, 0.001 < P < 0.01; *, 0.01 < P < 0.05, NS: not significant). B, HEK293 cells were transfected with plasmids coding for HA-FXR (wild type or mutant) and VP16, as negative control, or VP16-PGC1{alpha} (AA 1-400). Cells were treated (+) or not (–) with PMA (100 nM) for 15 min. Proteins were analyzed by Western blotting using FXR and VP16 antibodies. C, HEK293 cells were cotransfected for 12 h with TkpGL3-3xFXRE-pLuc (100 ng), pcDNA3-FXR (1 ng) (wild type or mutants), and pCDNA3 (1 ng) as negative control or full-length PGC1{alpha}. Cells were treated during 12 h with vehicle (white bars) or GW4064 (500 nM; gray bars) in the absence or presence of PMA (100 nM). Results are expressed in fold induction. Values are means ± SD (n = 3). Statistically significant differences between control and treated cells are indicated by asterisks [Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant, P > 0.05]. Ctr, Control; DMSO, dimethylsulfoxide; IB, immunoblotting; WT, wild type.

 
To confirm that FXR phosphorylation modulates this interaction in a cellular context, coimmunoprecipitation assays were performed using expression plasmids coding for VP16-PGC1{alpha} (AA 1-400) and hemagglutinin (HA)-FXR in HEK293 cells treated or not with PMA. Western blot analysis using a FXR antibody revealed that similar levels of FXR proteins were immunoprecipitated (Fig. 10BGo). PMA treatment clearly induced the recruitment of VP16-PGC1{alpha} (AA 1-400) by wild-type, but not mutant FXR protein, as revealed by Western blot analysis using a VP16 antibody (Fig. 10BGo).

Finally, the effect of PMA on PGC1{alpha}-induced FXR activation was investigated in a transactivation assay (Fig. 10CGo). PMA treatment increased the transcriptional activity of FXR, induced by GW4064, an effect that was enhanced in the presence of PGC1{alpha}, whereas the FXR S135AS154A mutant was insensitive to PMA stimulation and to PGC1{alpha} coactivation.

Overall, these data demonstrate that the FXR phosphorylation by PKC{alpha} increases its transcriptional activity by promoting recruitment of coactivators, such as PGC1{alpha}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
It has been previously reported that the transcription activity of several nonsteroidal nuclear receptors, including the retinoic acid receptor {alpha} (RAR{alpha}) (48), vitamin D3 receptor (49), peroxisomal proliferator-activated receptor (PPAR){alpha} (50, 51), pregnane X receptor (52), the orphan receptor TR2 (53, 54), and the retinoid-related orphan receptor {alpha}1 (55) is modulated by the PKC signaling pathway. However, whether FXR transcription activity is regulated by kinases had not been investigated before. In this study, we demonstrate, by using pharmacological PKC modulators and by overexpression of a constitutive active PKC{alpha} protein, that PKC activity is necessary for a maximal ligand-dependent induction of FXR transcription activity. By using kinase inhibitors, we show that the PKC, but not the PKA and PI3K, signaling pathway is implicated in the regulation of FXR target gene expression in response to its ligands. Indeed, PKC inhibitors diminished the expression of SHP and UGT2B4, which are up-regulated, respectively, via FXR/RXR heterodimers or FXR monomers (13, 15). PKC inhibition also prevented the repression of apoA-I, a negatively regulated FXR target gene. Based on these data, we investigated whether PKC could promote the transcription activity of FXR. By using PKC modulators in a transactivation assay, we showed that inhibition of the PKC signaling pathway by Rö 31-8220 and Gö-6976 decreases the transactivation of FXR on its classical IR1 response element, whereas treatment with PMA increases it. The effect of Gö-6976 (45) suggested that FXR transcription activity is regulated by Ca2+-dependent PKC isoforms such as PKC{alpha}, PKCβI, PKCβII, and PKC{gamma}. This up-regulation of FXR activity by PKC suggests that FXR, itself, or one of its coactivators could be phosphorylated by this kinase pathway. Indeed, the activity of coactivators such as SRCs, cAMP response element binding protein-binding protein/p300, PGC1{alpha}, and DRIP205 is also modulated by phosphorylation (56, 57, 58, 59). Moreover, a recent study demonstrated that SRC-3 is phosphorylated by PKC and that this phosphorylation leads to a cellular accumulation of SRC-3 and to an increase of estrogen receptor (ER)-dependent gene transcription (60). However, Yi et al. (59) showed that SRC-3 is phosphorylated by atypical PKCs, whereas we show that FXR transcription activity is modulated by calcium-dependent PKCs. Furthermore, SRC-3 has not been identified as FXR coactivator. Among the coactivators known to interact with FXR, DRIP205 was identified to be a substrate of PKC. However, the role of PKC phosphorylation on the coactivator function of DRIP205 has not been elucidated. Hence, it cannot be excluded that the regulation of FXR transcription activity by calcium-dependent PKCs may also involve the phosphorylation of coactivators or other partner proteins.

In this manuscript, we focused on the hypothesis that this cross talk between FXR and calcium-dependent PKC signaling could implicate phosphorylation of FXR. Indeed, in HepG2 cells, we demonstrated that endogenous FXR is a phosphoprotein and that PMA treatment induces the appearance of different phosphorylated FXR forms. These data suggested that PKC phosphorylates endogenous FXR probably on several residues. We also investigated whether Ca2+-dependent PKCs could phosphorylate FXR in vitro. Our data show clearly that FXR is phosphorylated in vitro by PKC{alpha} and PKCβI. Whether FXR is also regulated by other PKC classes or isoforms cannot be excluded and requires further study.

For the nuclear receptors RAR (48), vitamin D3 receptor (49), PPAR{alpha} (50, 51), and TR2 (54), the PKC-phosphorylated serine residues were localized in the DNA-binding domain and in the hinge region. Our data show that FXR is also phosphorylated by PKC in its DBD via residues S135 and S154. Mutation of these residues in alanine abolished PKC{alpha}CA-induced FXR transcriptional activity, indicating that S135 and S154 are required in the PKC{alpha}-dependent regulation of FXR activity. Phosphorylation of TR2 and PPAR{alpha}, by PKC in their DBD, favors their transcriptional activity (51, 54). In a very similar manner, calcium-dependent PKC phosphorylation also promotes FXR activity. However, a recent study showed that phosphorylation of a serine highly conserved in nuclear receptors, such as HNF4{alpha} and probably RAR{alpha}, RXR{alpha}, TRβ, and PPAR{alpha}, decreases their transcriptional activity (61). In contrast, replacement of the corresponding highly conserved S154 residue by alanine in the FXR protein led to a strong decrease of its basal activity.

The mechanism of action of FXR phosphorylation on its transcriptional activity was also investigated. FXR phosphorylation could modulate its DNA binding activity, its interaction with RXR or others partners such as coactivators and corepressors, and its subcellular localization. Our results show, by using a GFP-FXR fusion protein, that FXR is mainly present in the nucleus, and that PMA does not modulate this localization pattern. Moreover, in vitro phosphorylation of FXR did not increase its binding to its response element in an EMSA assay. Transfection experiments in HepG2 cells demonstrated that PMA increases the activity of GAL4-FXR fusion protein, confirming that PKC promotes the transcriptional activity of FXR in a FXRE-independent manner. Furthermore, this result indicates that the regulation by PKC is independent of the interaction between FXR and RXR. In addition, the fact that Rö31-8220 and Gö-6976 inhibit also monomeric FXR target genes, such as UGT2B4 and apoA-I, renders the hypothesis that FXR phosphorylation modulates its interaction with RXR very unlikely. Several studies revealed that phosphorylation of nuclear receptors modifies their interaction with coactivators. Indeed, phosphorylation of ER{alpha} in AF-1 domain is required for the recruitment of the coactivator p68 (62). Furthermore, phosphorylation of ER{alpha} and ERβ promotes interaction with the coactivators p160/SRC and cAMP response element binding protein-binding protein (63, 64, 65). Because PGC1{alpha} has been described to interact with the FXR DBD, the effect of FXR phosphorylation on the recruitment of PGC1{alpha} was investigated (20). Coimmunoprecipitation and GST-pull-down assays clearly showed that phosphorylation of the FXR-DBD promotes the recruitment of PGC1{alpha} to FXR. In a functional transactivation assay, overexpression of full-length PGC1{alpha} enhanced, in the presence of PMA, the transactivation of FXR induced by the GW4064. These results suggest that phosphorylation of FXR by PKC{alpha} enhances its transcriptional activity by increasing recruitment of cofactors such as PGC1{alpha}. The hypothesis that PGC1a phosphorylation could be implicated in this interaction is unlikely for several reasons. First, the FXR mutant did not recruit PGC1{alpha} in a PMA-dependent manner. Second, the GST pull-down assay showing PGC1{alpha} interaction was performed with a FXR DBD phosphorylated in vitro. Third, although it has been demonstrated that PGC1{alpha} is phosphorylated by p38 MAPK (58), so far, PGC1{alpha} has not been identified to be a PKC substrate.

During the last decade, the concept of selective nuclear receptor modulators (SNuRMs) emerged. The prototypical SNuRM is tamoxifen, which, as a selective estrogen receptor modulator, can activate or inhibit estrogen receptor action depending on the tissue. SNuRM-induced alterations in the conformation of the ligand-binding domains of nuclear receptors influence their abilities to interact with other proteins, such as coactivators and corepressors. However, recent evidences revealed that the cellular environment also plays a critical role in determining SNuRM biocharacteristics. For FXR, Dussault et al. (66) identified a selective bile acid receptor modulator (SBARM), which acts as an antagonist on I-BABP target gene expression and remains neutral on SHP expression. Based on the SNuRM concept, this selectivity in the regulation of FXR target genes could be partly explained by a differential interaction with cofactors. Our study showing the importance of phosphorylation on FXR activity adds a supplementary complexity to the molecular mechanism of SBARM action. The state of FXR phosphorylation, induced by PKC{alpha}, in various cellular environments could directly influence the ability of agonists to activate FXR and result in a SBARM effect.

Many reports have shown that bile acids modulate PKC pathways via different mechanisms. First, in the absence of DAG, bile acids increase PKC activity by facilitating its association with phospholipids (37). Second, bile acids stimulate phosphatidylinositol-specific phospholipase C and hence increase DAG formation (67). This present study shows that calcium-dependent PKCs regulate the ligand-dependent FXR activation. Consequently, we can hypothesize that FXR ligands, such as bile acids, could probably sensitize their own ligand effects on FXR by inducing PKC{alpha}-mediated FXR phosphorylation, demonstrating the existence of double converging FXR activation pathways by bile acids.

In summary, this study demonstrates, for the first time, that FXR is a phosphoprotein and the implication of the calcium-dependent PKC pathway in the regulation of FXR activity and its response to ligands. Moreover, we demonstrate that phosphorylation of FXR by PKC promotes PGC1{alpha} recruitment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
CDCA, Wortmannin, Ro 31-8220 methanolsulfonate salt, PKA inhibitor (PKI), and TRI-reagent were purchased from Sigma (Saint-Quentin, France). Gö6976 and PMA were from Calbiochem (Strasbourg, France) and Cell Signaling Technology (Danvers, MA), respectively. Human hepatoblastoma HepG2 and HEK293 cells were from American Type Culture Collection (Manassas, VA). JETPEI and Optifect transfection reagents were from PolyPlus Transfection (Illrich, France) and Invitrogen (Cergy-Pontoise, France), respectively. GW4064 was synthesized at GENFIT (Loos, France), according to Maloney et al. (6).

Cell Culture and Treatment
HepG2 and HEK293 cells were respectively grown in DMEM and modified Eagle’s medium supplemented with 10% fetal calf serum, streptomycin/penicillin, sodium pyruvate, glutamine, and nonessential amino acids (Life Technologies, Cergy-Pontoise, France) at 37 C, in a humidified 5% CO2 atmosphere. For mRNA analysis, HepG2 cells were treated in six-well plates at 60% confluence with either CDCA or GW4064 at the indicated concentration. For transactivation assays, with the various kinase inhibitors, cells were pretreated during 1 h before adding FXR agonists for the indicated time. For phosphorylation experiments, cells were treated with PMA for 10 min.

RNA Analysis
Total RNA was isolated from cells using TRI-Reagent according to the manufacturer’s instructions (Sigma, Saint-Quentin, France). For gene expression analysis, total RNA was reverse transcribed using random hexamer primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Paisley, Scotland). Real-time quantitative PCR analysis was performed using the LightCycler Technology (Roche Diagnostic, Meylan, France). For mRNA amplifications, 0.5 µl cDNA was added in 20 µl final volume to 2 µl of LightCycler DNA Master SYBR Green I mix (Roche Diagnostic), MgCl2 solution (3 mM), and forward and reverse oligonucleotide primers (100 nM). The PCR program consisted of a denaturing step at 95 C for 8 min followed by 40 cycles of 10 sec at 55 C, 10 sec at 95 C, and 15 sec at 72 C. Primers used were as follows: SHP forward, 5'-GGCTGGCAGTGCTGATTCAG-3'; reverse, 5'-TGGGGTGTGGCTGAGTGAAG-3'; UGT2B4 forward, 5'-ACTCTATATTAAGTATTTTGCTAA-3'; reverse, 5'-GGCTGAGATGAGTGTTACTAC-3'; apoA-I forward, 5'-TGAAAGCTGCGGTGCTGACC-3'; reverse, 5'-GTTCGCGCAGCTTGCTGAAG-3'; 36B4 forward, 5'-CATGCTCAACATCTCCCCCTTCTCC-3'; reverse, 5'-GGGAAGGTGTAATCCGTCTCCACAG-3'. Transcript levels were normalized to 36B4 mRNA levels.

Transient Transfection Assays
HepG2 or HEK293 cells were transfected using JETPEI reagent according to the manufacturer’s instructions in 24-well plates with 70 x 103 cells per well or 96-well plates with 40 x 103cells per well for dose response analysis. For 24-well plates, 100 ng of luciferase reporter plasmid was cotransfected with 20 ng of pRLnull Renilla reporter plasmid as an internal control of transfection, 20 ng of pcDNA3hFXR or pcDNA3 (Invitrogen, Leek, The Netherlands), and 20 ng of pSG5mRXR{alpha}, or pSG5 (Stratagene, La Jolla, CA). cells were incubated 12 h after transfection with medium containing 2% Ultroser SF serum (Life Technologies, Cergy-Pontoise, France) in the presence of ligand. For 96-well plates, 100 ng luciferase reporter plasmid was cotransfected with 1 ng of pcDNA3-FXR in the presence or not of the expression plasmid coding for constitutive active PKC{alpha}. Cells were incubated 12 h after transfection with DMEM without serum. Luciferase activity was assayed using a TR717 microplate luminometer (Applied Biosystems, Courtaboeuf, France).

For in cell phosphorylation experiments, 106 HEK293 cells were seeded in 10-cm dishes 24 h before the transfection. Using the optifect reagent, 30 µg of pCDNA3FXR (wild type or mutant) was transfected according to the manufacturer’s instructions.

Production and Purification of Protein
Production and purification of FXR fragments were performed using the pGEX 5X1 cloning vector (GE Healthcare, Orsay, France) and expressed in Escherichia coli BL21 bacteria. The expression of the fusion proteins was induced for 4 h with 0.5 mM isopropyl-β-D-thiogalactopyranoside at 30 C. Bacterial pellets were resuspended in lysis buffer containing 1xPBS, 1 mM dithiothreitol (DTT), 1 mM benzamidine, 0.125 mg/ml, and 0.1% Nonidet P-40 (NP-40). Cell suspensions were sonicated and centrifuged at 10,000 x g for 30 min. Supernatants were incubated with glutathione-Sepharose 4B (GE Healthcare) for 3 h at 4 C. After incubation, the beads were washed in lysis buffer. Proteins were quantified by SDS-PAGE and Coomassie blue staining. GST-FXR fragment bound beads were incubated with factor Xa (10 U/500 µg protein) for 4 h, at room temperature. The factor Xa (GE Healthcare) was removed using p-aminobenzamidine agarose resin. Proteins were quantified by SDS-PAGE and Coomassie blue staining.

The phosphoproteins were purified from HepG2 cells according to the manufacturer’s instructions (QIAGEN, Courtaboeuf, France) as described previously (47). The different fractions were separated by SDS-PAGE and then immunoblotted with an anti-FXR antibody (Perseus Proteomics, Tokyo, Japan). For treatment with CIP, 1 mg of proteins was incubated with 100 U for 1 h at 37 C.

In Vitro Phosphorylation Assays
Phosphorylation assay of the purified FXR protein was performed using purified calcium-dependent PKC isoforms (Euromedex, Souffelweyersheim, France) according to the manufacturer’s instructions. FXR fragments were incubated with purified PKC, in buffer containing 20 mM HEPES, 10 mM MgCl2, 0.1 mM CaCl2, 1 mM ATP, the lipid activator, and phosphatase inhibitors. After 30 min at 30 C, the proteins were analyzed by SDS-PAGE. The phosphorylation of FXR fragments were revealed after blotting on nitrocellulose membranes and immunodetection with antiphosphoserine or antiphosphothreonine antibodies (Cell Signaling Technology).

Two-Dimensional Gel Analysis
Proteins (100 µg) extracted from HepG2 cells in buffer containing 7 M urea, 2 M thiourea, 4% 3-(cholamidopropyl)dimethyl ammonio-1-propane sulfonic acid, 40 mg DTT, were loaded on Readystrip IPG strips, pH 5-8, 17 cm. Isoelectric focusing was carried out on PROTEAN IEF cell (Bio-Rad, Marne la coquette, France) consisting of five phases of stepped voltages from 250 V to 10,000 V.

Before two-dimensional electrophoresis, gel strips were equilibrated in buffer containing 6 M urea, 2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH 8.8), 20% glycérol reduced with 30 mM DTT and subsequently with 4.5% iodoacetamide. The gel strips were applied to a 10% SDS-PAGE gel, and gels were run at 25 mA/gel for 10 min, increasing to 50 mA/gel.

Proteins were blotted on nitrocellulose membranes, stained with Sypro ruby protein blot (Molecular Probes, Inc., Eugene, OR) and subjected to immunodetection using an anti-FXR antibody (Perseus Proteomics).

GST Pull-Down Assay
GST-FXR DBD fusion protein was expressed in BL21 as described above. Bacterial pellet resuspended in lysis buffer containing 1x PBS, 1 mM DTT, 1 mM benzamidine, 0.125 mg/ml, and 0.1% NP-40. Cell suspensions were sonicated and centrifuged at 10,000 x g for 30 min. Supernatants were incubated with glutathione-Sepharose 4B for 3 h at 4 C. After incubation, the beads were washed in lysis buffer, and proteins were quantified by SDS-PAGE and Coomassie blue staining.

GST-FXR DBD fusion protein (20 µg) immobilized on beads was phosphorylated or not, as described above. It were incubated with 5 µg of His-PGC1{alpha} (AA 1-400) in GST-binding buffer [15 mM Tris HCl (pH 8), 15% glycerol, 0.15 mM EDTA, 0.0375% NP-40, 112.5 mM KCl, 0.25 mg/ml BSA, 1 mM DTT, 1 mM benzamidine] for 2 h at room temperature. Bound proteins were washed four times with washing buffer (GST-binding buffer containing 0.05% NP-40 and without BSA) and samples were denaturated in Laemmli at 94 C for 10 min. Proteins were separated by SDS-PAGE and blotted on a nitrocellulose membrane. GST-FXR DBD proteins were visualized by Western blot analysis using a GST antibody (Interchim, Montluçon, France). The His-PGC1{alpha} (AA 1-400) was probed with the anti-penta-His horseradish peroxidase conjugate antibody (QIAGEN). Signal corresponding to His-PGC1{alpha} recruitment was quantified by densitometry using Bio-Rad Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA).

Coimmunoprecipitation
HEK293 cells were cotransfected with 15 µg of plasmid coding for HA-FXR (wild type or mutant) or VP16-PGC1{alpha} (AA 1-400). Cells were treated 24 h later or not with PMA (100 nM) for 15 min. Cells were resuspended in lysis buffer [150 mM NaCl, 50 mM Tris HCl (pH 8), 1% NP-40, 1 mM DTT, protease inhibitors, phosphatase inhibitors] and incubated for 2 h at 4 C. Samples were cleared by centrifugation, and protein concentrations were determined. Equivalent amounts of proteins were precleared for 1 h with protein G agarose beads. Mouse anti-HA (12CA5) was added to the samples and incubated overnight at 4 C in the presence of protein G agarose beads. The beads were washed five times with cold lysis buffer, resuspended in Laemmli, and heated for 10 min at 95 C. Proteins were separated by SDS-PAGE, blotted on a nitrocellulose membrane, and revealed with FXR and VP 16 antibodies.

EMSAs
Gel shift assays were performed as previously described (68) using [{gamma}-32P]ATP-labeled oligonucleotide probe containing the wild-type or mutated I-BABP IR-1 FXR binding site, 1 µg of the purified FXR protein phosphorylated in vitro using PKC{alpha}, and hRXR{alpha} protein synthesized in vitro using the TNT Quick Coupled Transcription/Translation system (Promega Corp., Madison, WI).

Fluorescence Microscopy
HEK293 cells were transfected using GFP-FXR for 24 h. Cells were treated or not with PMA (100 nM) for 2 h and washed in PBS, fixed in 4% paraformaldehyde. After two washes in PBS, cells on coverslips were mounted with a drop of Vectashield (Vector Laboratories, Inc., Burlingame, CA). Fluorescence microscopy was performed on an Olympus IX81 (Olympus Corp., Lake Success, NY).


   ACKNOWLEDGMENTS
 
We thank K. Bertrand and the Chemistry Department of GENFIT for the synthesis of the GW4064 compound. We thank Professor Parker for providing the constitutive active PKC{alpha} expression vectors.


   FOOTNOTES
 
This work was supported by grants from ANR (No. A05056GS) and HEPADIP (No. 018734). R.G. is recipient of a Conventions Industrielles de Formation par la Recherche training grant from Association Nationale de la Recherche Technique and the Ministère de la Recherche.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 28, 2008

Abbreviations: AA, Amino acids; apo, apolipoprotein; CDCA, chenodeoxycholic acid; CIP, calf intestinal phosphatase; DAG, diacylglycerol; DBD, DNA-binding domain; DRIP, vitamin D receptor-interacting protein; DTT, dithiothreitol; ER, estrogen receptor; GFP, green fluorescent protein; FXR, farnesoid X receptor; FXRE, FXR response element; GST, glutathione-S-transferase; HA, hemagglutinin; HDL, high-density lipoprotein; HEK, human embryonic kidney; I-BABP, ileal bile acids-binding protein; NP-40, Nonidet P-40; PGC-1{alpha}, PPAR{gamma} coactivator 1{alpha}; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PKC{alpha}CA constitutive active PKC{alpha}; PKI, PKA inhibitor; PMA, phorbol 12-myristate 13-acetate; PPAR, peroxisomal proliferator-activated receptor; PRMT, protein arginine methyl-transferase; RAR{alpha}, retinoic acid receptor {alpha}; RXR, retinoid X receptor; SBARM, selective bile acid receptor modulator; SHP, small heterodimer partner; SnuRM, selective nuclear receptor modulator; SRC, steroid receptor coactivator; TG, triglyceride; UGT2B4, UDP-glucuronosyltransferase 2B4.

Received for publication March 19, 2008. Accepted for publication August 19, 2008.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   SHP  |  FXRα
Coregulators:   PGC-1
Ligands:   GW4064



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P. B. Hylemon, H. Zhou, W. M. Pandak, S. Ren, G. Gil, and P. Dent
Bile acids as regulatory molecules
J. Lipid Res., August 1, 2009; 50(8): 1509 - 1520.
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