The retinoic acid-related orphan receptor ␣ (ROR␣) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of ROR␣. We have now solved the x-ray crystal structure of the ligand binding domain of ROR␣ in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the ROR␣ ligand binding domain, namely NH-Gln 289 , NH-Tyr 290 , and NH1-Arg 370 . When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of ROR␣. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of ROR␣ in vivo.The group of retinoic acid-related orphan nuclear receptors (ROR) 1 is encoded by three different genes (␣, ␤, and ␥) (1). ROR␣ has been implicated in numerous age-related phenotypes such as atherosclerosis, cerebellar atrophy, immunodeficiency, and bone metabolism (2). ROR␣ was still considered an orphan receptor until we recently reported the first crystal structure of the ROR␣ LBD. It had revealed a ligand that was unexpectedly present, namely cholesterol (3). We also had shown that the transcriptional activity of ROR␣ could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. This has led to the hypothesis that ROR␣ could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases. Despite the relatively high homology between ROR␣ LBD and ROR␤ LBD (63%), cholesterol seems not to be a ligand for the ROR␤ isoform, as reported recently by . This indicates a possible distinct function for ROR␤ and ROR␣. An inspection of the x-ray structure of the complex between ROR␣ LBD and cholesterol had shown that in the hydrophilic part of the LBP, there is space for a substituent attached to the hydroxy group of cholesterol, if water molecules are displaced (3). The presence of three arginines (Arg 292 , Arg 370 , and Arg 367 ) and of two free backbone amide nitrogens (NH-Gln 289 and NH-Tyr 290 ) strongly suggested a negatively charged substituent with at least two hydrogen-bond acceptor functionalities. Docking studies led to the prediction that cholesterol sulfate should have higher affinity than cholest...

The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. No X-ray structure of RORalpha has been described so far, and no ligand has been identified. We describe the first crystal structure of the ligand binding domain (LBD) of RORalpha, at 1.63 A resolution. This structure revealed a ligand present in the ligand binding pocket (LBP), which was identified by X-ray crystallography as cholest-5-en-3beta-ol (cholesterol). Moreover, RORalpha transcriptional activity could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. These findings suggest that RORalpha could play a key role in the regulation of cholesterol homeostasis and thus represents an important drug target in cholesterol-related diseases.

The nuclear orphan receptor human estrogen receptor-related receptor (ERR)-alpha is implicated in bone metabolism. We studied the effect of ERRalpha silencing in human mesenchymal stem cells (hMSCs) during osteoblastogenesis. We found that ERRalpha silencing led to an increase of bone sialoprotein and a decrease of osteopontin mRNA levels, suggesting enhanced osteoblastic differentiation. This was confirmed by an increased ability of hMSCs to deposit calcium. Concomitantly, knockdown of ERRalpha inhibited adipogenesis, resulting in a decrease in adipocyte number and adipocyte marker gene expression. In line with a negative role of ERRalpha in bone metabolism, we found that adult female and male ERRalpha-deficient mice displayed a moderate increase in femoral cancellous bone volume and density. Osteoblast surface was increased and marrow fat volume decreased in these animals. Furthermore, ERRalpha-deficient osteoblasts displayed increased differentiation properties in vitro in line with our observations in hMSCs. In summary, we identified a role for ERRalpha in bone mass regulation by affecting osteoblastic differentiation.

No abstract

No abstract