Synergistic regulation of the mouse orphan nuclear receptor SHP gene promoter by CLOCK-BMAL1 and LRH-1

Small heterodimer partner (SHP; NR0B2) is an orphan nuclear receptor and acts as a repressor for wide variety of nuclear hormone receptors. We demonstrated here that mouse SHP mRNA showed a circadian expression pattern in the liver. Transient transfection of the mSHP promoter demonstrated that CLOCK-BMAL1, core circadian clock components, bound to E-box (CACGTG), and stimulated the promoter activity by 4-fold. Liver receptor homologue-1 (LRH-1; NR5A2) stimulated the mSHP promoter, and CLOCK-BMAL1 synergistically enhanced the LRH-1-mediated transactivation. Interestingly, SHP did not affect the CLOCK-BMAL1-mediated promoter activity, but strongly repressed the synergistic activation of CLOCK-BMAL1 and LRH-1. Furthermore, in vitro pull-down assays revealed the existence of direct protein-protein interaction between LRH-1 and CLOCK. In summary, this study shows that CLOCK-BMAL1, LRH-1 and SHP coordinately regulate the mSHP gene to generate the circadian oscillation. The cyclic expression of mSHP may affect daily activity of other nuclear receptors and contribute to circadian liver functions.     

Molecular dynamics simulations of human LRH-1: the impact of ligand binding in a constitutively active nuclear receptor

The liver receptor homologue 1 (LRH-1 (NR5A2)) belongs to the orphan nuclear receptor family, indicating that initially no ligand was known. Although recent studies have shown that ligand binding can be obtained, the biological relevance remains elusive. Here, we modify the observed X-ray ligand into a biologically more significant phospholipid (phosphatidylserine, PS) present in human, to study, by molecular dynamics (MD) simulations, the impact of the ligand on the receptor and the interaction with different cofactor peptides. Furthermore, we characterize the interactions between receptor and the cofactor peptides of DAX-1 (NR0B1), Prox1 and SHP LXXLL box 1 and 2 (NR0B2) in terms of specificity. Our MD simulation results show different interaction patterns for the SHP box2 compared to DAX-1, PROX1 and SHP box1. SHP box2 shows specific interactions at its more C-terminal end while the other investigated peptides show specific interactions at several positions but particularly at the +2 site. The peptide +2 side chain interacts with a charged amino acid of the receptor, in hLRH-1 Asp372. Together with the charge clamp residues Arg361 and Glu534, Asp372 forms a triangle shaped charge clamp responsible for peptide orientation and increased affinity. The binding of the PS ligand causes no overall structural changes of the receptor but affects the interactions with cofactor peptides. The cofactor peptides from SHP decrease its interaction with the receptor upon ligand binding while DAX-1 and PROX1 are unchanged or increase. The diverse ligand binding response of the cofactor provides an opportunity for drug design with the possibility to create agonist ligands to modify cofactor interaction.     

Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1

The orphan nuclear receptors small heterodimer partner (SHP) and dosage-sensitive sex-reversal adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1 (DAX-1) contain extra amino acids between helices H6 and H7 of LBD, and here we investigated a possible role of these additional amino acids. Transient transfection assay demonstrated that, in contrast to wild type, in mutant SHP Delta128-139 deletion of 12 extra amino acids in H6-H7 failed to repress the transactivity of orphan nuclear receptors such as estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Interestingly, yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that wild-type and SHP Delta128-139 have similar abilities to interact with estrogen receptor-related receptor-gamma, hepatocyte nuclear factor 4alpha, and constitutive androstane receptor. Unexpectedly, in wild-type DAX-1 and mutant DAX-1 Delta338-362, deletion of 25 extra amino acids in H6-H7 had no significant difference in the interaction and repression of steroidogenic factor 1 transactivation. Mutant SHP that contains DAX-1 extra amino acids or polyalanine stretch in H6-H7 showed indistinguishable pattern of repression from wild-type SHP. Interestingly, the swapped SHP mutant with DAX-1 extra amino acids interacted with EID-1 (E1A-like inhibitor of differentiation 1), which is characterized as an SHP-interacting corepressor. However, interaction between SHP Delta128-139 and EID-1 was significantly diminished. Moreover, SHP-mediated repression of constitutive androstane receptor transactivation was significantly released by down-regulation of EID-1 expression with EID-1 small interfering RNA. The present study suggests that H6-H7 loop regions of SHP and DAX-1 play a different role in the repression of nuclear receptor transactivation.     

The atypical orphan nuclear receptor DAX-1 interacts with orphan nuclear receptor Nur77 and represses its transactivation

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on the X chromosome, gene 1) (NROB1) is an atypical member of the nuclear receptor family, which lacks the classical zinc finger DNA binding domain and acts as a coregulator of a number of nuclear receptors. In this study, we have found that DAX-1 is a novel coregulator of the orphan nuclear receptor Nur77 (NR4A1). We demonstrate that DAX-1 represses the Nur77 transactivation by transient transfection assays. Specific interaction between Nur77 and DAX-1 was detected by coimmunoprecipitation, yeast two-hybrid, and glutathione-S-transferase pull-down assays. The ligand binding domain of DAX-1 and the activation function-2 domain of Nur77 were determined as the direct interaction domains between DAX-1 and Nur77. In vitro competition binding assay showed that DAX-1 repressed Nur77 transactivation through the competition with steroid receptor coactivator-1 for the binding of Nur77. Moreover, DAX-1 repressed Nur77- and LH-dependent increase of cytochrome P450 protein 17 promoter activity in transient transfection assays. Furthermore, Nur77-mediated transactivation was significantly increased by down-regulation of DAX-1 expression with DAX-1 small interfering RNA in testicular Leydig cell line, K28. LH treatment induced a transient increase in Nur77 mRNA, whereas LH repressed DAX-1 expression in a time- and dose-dependent manner in K28 cells. In addition, immunohistochemical analysis showed the expression of Nur77 in mouse testicular Leydig cells. These results suggest that DAX-1 acts as a novel coregulator of the orphan nuclear receptor Nur77, and that the DAX-1 may play a key role in the regulation of Nur77-mediated steroidogenesis in testicular Leydig cells.     

DAX-1, an unusual orphan receptor at the crossroads of steroidogenic function and sexual differentiation

The unusual orphan member of the nuclear hormone receptor superfamily DAX-1 (NR0B1) owes its name to its double role in human pathology. On one side, duplications in Xp21, containing the DAX-1 gene, cause phenotypic sex reversal in XY individuals. On the other side, DAX-1 gene mutations are responsible for adrenal hypoplasia congenita, invariably associated with hypogonadotropic hypogonadism. DAX-1 functions as a global negative regulator of steroid hormone production by repressing the expression of multiple genes involved in the steroidogenic pathway. Here we review the mechanism of DAX-1 function in adrenal and gonadal differentiation, with special emphasis on recent results showing the critical role of DAX-1 protein misfolding in the pathogenesis of adrenal hypoplasia congenita.