Hyperphosphorylation of the retinoid X receptor alpha by activated c-Jun NH2-terminal kinases.

The nuclear receptor mouse retinoid X receptor alpha (mRXRalpha) was shown to be constitutively phosphorylated in its NH2-terminal A/B region, which contains potential phosphorylation sites for proline-directed Ser/Thr kinases. Mutants for each putative site were generated and overexpressed in transfected COS-1 cells. Constitutively phosphorylated residues identified by tryptic phosphopeptide mapping included serine 22 located in the A1 region that is specific to the RXRalpha1 isoform. Overexpression and UV activation of the stress-activated kinases, c-Jun NH2-terminal kinases 1 and 2 (JNK1 and JNK2), hyperphosphorylated RXRalpha, resulting in a marked decrease in its electrophoretic mobility. This inducible hyperphosphorylation involved three residues (serines 61 and 75 and threonine 87) in the B region of RXRalpha and one residue (serine 265) in the ligand binding domain (E region). Binding assays performed in vitro with purified recombinant proteins demonstrated that JNKs did not interact with RXRalpha but bound to its heterodimeric partners, retinoic acid receptors alpha and gamma (RARalpha and RARgamma). Hyperphosphorylation by JNKs did not affect the transactivation properties of either RXRalpha homodimers or RXRalpha/RARalpha heterodimers in transfected cultured cells.     

Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.     

Analysis of protein dimerization and ligand binding of orphan receptor HNF4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha) (NR2A1), an orphan member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer even though it is very similar in amino acid sequence to retinoid X receptor alpha (RXRalpha), which heterodimerizes readily with other receptors. Here, experimental analysis of residues involved in protein dimerization and studies on a reported ligand for HNF4alpha are combined with a structural model of the HNF4alpha ligand-binding domain (LBD) (residues 137 to 384). When K300 (in helix 9) and E327 (in helix 10) of HNF4alpha1 were converted to the analogous residues in RXRalpha (E390 and K417, respectively) the resulting construct did not heterodimerize with the wild-type HNF4alpha, although it was still able to form homodimers and bind DNA. Furthermore, the double mutant did not heterodimerize with RXR or RAR but was still able to dimerize in solution with an HNF4alpha construct truncated at amino acid residue 268. This suggests that the charge compatibility between helices 9 and 10 is necessary, but not sufficient, to determine dimerization partners, and that additional residues in the HNF4alpha LBD are also important in dimerization. The structural model of the HNF4alpha LBD and an amino acid sequence alignment of helices 9 and 10 in various HNF4 and other receptor genes indicates that a K(X)(26)E motif can be used to identify HNF4 genes from other organisms and that a (E/D(X)(26-29)K/R) motif can be used to predict heterodimerization of many, but not all, receptors with RXR. In vitro analysis of another HNF4alpha mutant construct indicates that helix 10 also plays a structural role in the conformational integrity of HNF4alpha. The structural model and experimental analysis indicate that fatty acyl CoA thioesters, the proposed HNF4alpha ligands, are not good candidates for a traditional ligand for HNF4alpha. Finally, these results provide insight into the mechanism of action of naturally occurring mutations in the human HNF4alpha gene found in patients with maturity onset diabetes of the young 1 (MODY1).     

The nuclear receptor coactivator PGC-1alpha exhibits modes of interaction with the estrogen receptor distinct from those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1alpha co-regulator (residues 91-408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1alpha to ER. Anisotropy measurements revealed that the affinity of this PGC-1alpha fragment for human ERalpha and beta was fairly strong in the presence of estradiol (approximately 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-191-408 exhibited ligand-independent interactions with ER, particularly with ERbeta (Kd approximately 30 nM). Competition experiments of the complex between ERalpha and fluorescently labelled PGC-1 91-408 with unlabelled SRC-1 570-780 showed that PGC-1 91-408 was an efficient competitor of SRC-1 570-780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERalpha, SRC-1 570-780 and PGC-1 91-408. GST-pull-down experiments with deletion mutants of ERalpha revealed that the constitutive binding of PGC-1 91-408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1alpha confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633-675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.     

Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.     

Peroxisome proliferator-activated receptor-gamma coactivator-1alpha activation of CYP7A1 during food restriction and diabetes is still inhibited by small heterodimer partner

Cholesterol 7alpha-hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic pathway of hepatic bile acid biosynthesis from cholesterol. During fasting and in type I diabetes, elevated levels of peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1alpha) induce expression of the Cyp7A1 gene and overexpression of PGC-1alpha in hepatoma cells stimulates bile acid synthesis. Using Ad-PGC-1alpha-RNA interference to induce acute disruption of PGC-1alpha in mice, here we show that PGC-1alpha is necessary for fasting-mediated induction of CYP7A1. Co-immunoprecipitation and promoter activation studies reveal that the induction of CYP7A1 is mediated by direct interaction between PGC-1alpha and the AF2 domain of liver receptor homolog-1 (LRH-1). In contrast, the very similar PGC-1beta could not substitute for PGC-1alpha. We also show that transactivation of PGC-1alpha and LRH-1 is repressed by the small heterodimer partner (SHP). Treatment of mice with GW4064, a synthetic agonist for farnesoid X receptor, induced SHP expression and decreased both the recruitment of PGC-1alpha to the Cyp7A1 promoter and the fasting-induced expression of CYP7A1 mRNA. These data suggest that PGC-1alpha is an important co-activator for LRH-1 and that SHP targets the interaction between LRH-1 and PGC-1alpha to inhibit CYP7A1 expression. Overall, these studies provide further evidence for the important role of PGC-1alpha in bile acid homeostasis and suggest that pharmacological targeting of farnesoid X receptor in vivo can be used to reverse the increase in CYP7A1 associated with adverse metabolic conditions.     

In vitro farnesoid X receptor ligand sensor assay using surface plasmon resonance and based on ligand-induced coactivator association

Ligand binding to nuclear receptors leads to a conformational change that increases the affinity of the receptors to coactivator proteins. We have developed a ligand sensor assay for farnesoid X receptor (FXR) in which the receptor–coactivator interaction can be directly monitored using surface plasmon resonance biosensor technology. A 25-mer peptide from coactivator SRC1 containing the LXXLL nuclear receptor interaction motif was immobilized on the surface of a BIAcore sensor chip. Injection of the FXR ligand binding domain (FXRLBD) with or without the most potent natural ligand, chenodeoxycholic acid (CDCA), over the surface of the chip resulted in a ligand- and LXXLL motif-dependent interaction. Kinetic analysis revealed that CDCA and its conjugates decreased the equilibrium dissociation constant (K_d) by 8–11-fold, indicating an increased affinity. Using this technique, we found that a synthetic bile acid sulfonate, 3,7-dihydroxy-5β-cholane-24-sulfonate, which was inactive in a FXR response element-driven luciferase assay using CV-1 cells, caused the most potent interaction, comparable to the reaction produced by CDCA. This method provides a rapid and reliable in vitro ligand assay for FXR. This kinetic analysis-featured technique may be applicable to mechanistic studies.     

Consumption of soy protein isolate reduces hepatic SREBP-1c and lipogenic gene expression in wild-type mice, but not in FXR-deficient mice

We examined to determine whether hepatic gene expression is affected in mice in which blood lipid levels remain unchanged fed soy protein isolate (SPI) for a short time. We also examined SPI-mediated effects in farnesoid X receptor (FXR)-deficient mice. Compared with casein, SPI affected the expression of various hepatic genes related to lipid metabolism in the wild-type mice. No effects of SPI were observed in the FXR-deficient mice, suggesting the importance of FXR. Hepatic peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) gene expression was reduced by SPI, and this might be associated with a decrease in FXR expression. Decreased FXR led to decreased expression of its target, the bile-salt export pump necessary for bile acid secretion and dietary lipid absorption. The earliest response to SPI was a decrease in hepatic sterol regulatory element-binding protein (SREBP)-1c mRNA, on day 3. SPI activated hepatic adenosine monophosphate-activated protein kinase (AMPK), which can lead to a reduction in SREBP-1c mRNA. These data indicate the importance of SREBP-1c and PGC-1α/FXR in SPI-mediated alterations in hepatic gene expression.     

Evidence for a novel cardiac-enriched retinoid X receptor partner.

Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.