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Crystal Structure of the Human RORα Ligand Binding Domain in Complex with Cholesterol Sulfate at 2.2 Å

2004; Elsevier BV; Volume: 279; Issue: 14 Linguagem: Inglês

10.1074/jbc.m400302200

1083-351X

Joerg Kallen, Jean-Marc Schlaeppi, Francis Bitsch, Isabelle Delhon, Brigitte Fournier,

Nuclear Receptors and Signaling

The retinoic acid-related orphan receptor α (RORα) 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 RORα. We have now solved the x-ray crystal structure of the ligand binding domain of RORα 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 RORα ligand binding domain, namely NH-Gln289, NH-Tyr290, and NH1-Arg370. 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 RORα. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORα in vivo. The retinoic acid-related orphan receptor α (RORα) 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 RORα. We have now solved the x-ray crystal structure of the ligand binding domain of RORα 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 RORα ligand binding domain, namely NH-Gln289, NH-Tyr290, and NH1-Arg370. 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 RORα. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORα in vivo. The group of retinoic acid-related orphan nuclear receptors (ROR) 1The abbreviations used are: ROR, retinoic acid-related orphan nuclear receptor; LBD, ligand binding domain; LBP, ligand binding pocket; ESI-MS, electrospray ionization mass spectrometry; DSC, differential scanning calorimetry. 1The abbreviations used are: ROR, retinoic acid-related orphan nuclear receptor; LBD, ligand binding domain; LBP, ligand binding pocket; ESI-MS, electrospray ionization mass spectrometry; DSC, differential scanning calorimetry. is encoded by three different genes (α, β, and γ) (1Jetten A.M. Kurebayashi S. Ueda E. Prog. Nucleic Acid Res. Mol. Biol. 2001; 69: 205-247Google Scholar). RORα has been implicated in numerous age-related phenotypes such as atherosclerosis, cerebellar atrophy, immunodeficiency, and bone metabolism (2Jarvis C.I. Staels B. Brugg B. Lemaigre-Dubreuil Y. Tedgui A. Mariani J. Mol. Cell. Endocrinol. 2002; 186: 1-5Google Scholar). RORα was still considered an orphan receptor until we recently reported the first crystal structure of the RORα LBD. It had revealed a ligand that was unexpectedly present, namely cholesterol (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). We also had shown that the transcriptional activity of RORα could be modulated by changes in intracellular cholesterol level or mutation of residues involved in cholesterol binding. This has led to the hypothesis that RORα 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 RORα LBD and RORβ LBD (63%), cholesterol seems not to be a ligand for the RORβ isoform, as reported recently by Stehlin-Gaon et al. (4Stehlin-Gaon C. Willmann D. Zeyer D. Sanglier S. Van Dorsselaer A. Renaud J-P. Moras D. Schüle R. Nat. Struct. Biol. 2003; 10: 820-825Google Scholar). This indicates a possible distinct function for RORβ and RORα. An inspection of the x-ray structure of the complex between RORα 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 (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). The presence of three arginines (Arg292, Arg370, and Arg367) and of two free backbone amide nitrogens (NH-Gln289 and NH-Tyr290) 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 cholesterol, and might be, because of its excellent fit and optimized interactions, the actual natural ligand of RORα (instead of cholesterol itself). Here, we present the x-ray crystal structure of the RORα LBD-cholesterol sulfate complex. We show a comparison with the x-ray structure of the RORα LBD-cholesterol complex, which suggests that cholesterol sulfate has a higher affinity than cholesterol. Indeed, as shown by mass spectrometry analysis, the exchange of cholesterol with cholesterol sulfate is practically irreversible under the conditions used. In addition, DSC analysis revealed that cholesterol sulfate increased the phase transition temperature for RORα LBD by 9 °C, relative to cholesterol. We also show that cholesterol sulfate shows increased (versus cholesterol) transcriptional activation, which is reduced by a point mutation that affects the binding of cholesterol sulfate more than that of cholesterol. We speculate that cholesterol sulfate plays a role in the regulation of RORα in vivo. RORα LBD Protein Preparation for Crystallization and ESI-MS Exchange Experiment—The RORα LBD protein (residues 271–523, flanked by an N-terminal hexa-His tag and a PreScission™ cleavage site) was expressed in the baculovirus system (Sf-9 cells) and purified as described previously (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). The exchange of cholesterol by cholesterol sulfate was done at 37 °C and confirmed by ESI-MS-analysis as reported previously (5Bitsch F. Aichholz R. Kallen J. Geisse S. Fournier B. Schlaeppi J-M. Anal. Biochem. 2003; 323: 139-149Google Scholar). Briefly, cholesterol sulfate was dissolved at 50 mm in Me2SO and added at 1.0 mm final concentration to the (His)6RORα LBD271–523 solution at 73 μm. The resulting solution was incubated overnight at 37 °C and further purified by size exclusion chromatography on an SPX75 column before concentration to 17.6 mg/ml for crystallization trials. MS determination of the native complex was done as described previously (5Bitsch F. Aichholz R. Kallen J. Geisse S. Fournier B. Schlaeppi J-M. Anal. Biochem. 2003; 323: 139-149Google Scholar). A control experiment was done by incubating the same amount of RORα LBD protein with 5% Me2SO under identical conditions. The protein concentration was ∼15 μm in 50 mm AcONH4, pH 7.0. Both spectra were recorded under identical conditions with Vc = 20 volts. Differential Scanning Calorimetry—The RORα LBD complexes with cholesterol and cholesterol sulfate, respectively, were obtained as described above. After the exchange experiment, the excess of cholesterol sulfate was removed on 5-ml HiTrap desalting columns. Protein concentration was determined by high pressure liquid chromatography-UV detection (214 nm). The final protein concentration following ligand exchange was 20 μm. DSC scans were obtained using a MicroCal VP-capDSC system (MicroCal, LLC, Northampton, MA) at a scan rate of 250 °C/h. Crystallization, Data Collection, and Structure Determination— Crystals were obtained at 4 °C by the vapor diffusion method in 2-μl hanging drops containing equal volumes of protein (17.6 mg/ml) and crystallization buffer (0.2 m MgCl2, 16% w/v polyethylene glycol 4000, 0.1 m Tris HCl, pH 8.5). The crystal form obtained was similar to the one for the complex with cholesterol (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). Diffraction data at 100 K were collected at the Swiss Light Source (beamline X06SA) using a Marre-search CCD detector and an incident monochromatic x-ray beam with 0.9200-Å wavelength. In total, 226 images were collected with 1.0° rotation each, using an exposure time of 9 s/frame and a crystal-to-detector distance of 150 mm. Raw diffraction data were processed and scaled with the HKL program suite version 1.96.1 (6Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar). The estimated B-factor by Wilson plot analysis is 32.9 Å2. The structure was determined using as starting model the coordinates of the complex RORα LBD-cholesterol refined to 1.63-Å resolution (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). The program REFMAC version 5.0 (7Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar, 8Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar) was used for refinement. Bulk solvent correction, an initial anisotropic B factor correction, and restrained isotropic atomic B-factor refinement were applied. The refinement target was the maximum likelihood target using amplitudes. No σ cut-off was applied on the structure factor amplitudes. Cross-validation was used throughout refinement using a test set comprising 5.0% (829) of the unique reflections. Water molecules were identified with the program ARP/wARP (7Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar, 9Lamzin V.S. Perrakis A. Wilson K.S. Rossmann M.G. Arnold E. International Tables for Crystallography. F. Kluwer Academic Publishers, Norwell, MA2001: 720-722Google Scholar) and selected based on difference peak height (greater than 3.0 σ) and distance criteria. Water molecules with temperature factors greater than 70 Å2 were rejected. The program O version 7.0 (10Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar) was used for model rebuilding, and the quality of the final refined model was assessed with the programs PROCHECK version 3.3 (11Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Cryst. 1993; 26: 283-291Google Scholar) and REFMAC version 5.0 (7Collaborative Computational Project Number 4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Google Scholar, 8Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar). Crystal data, data collection, and refinement statistics are shown in Table I.Table ICrystallographic summaryDiffraction data Space groupP21 Unit cell dimensionsa = 54.4 Åb = 49.9 Åc = 60.7 Åβ = 97.8° Resolution range20.0—2.2 Å (2.28—2.20 Å) No. of observations57,993 No. of unique reflections16,541 〈I/σ(I)〉16.2 Rsym on intensitiesaRsym = ΣIavg — II/ΣII0.079 Completeness99.7% (99.4%)Refinement Resolution range20.0—2.20 Å RcrystbRcryst = ΣFP — FPcalc/ΣFP, where FP and FPcalc are observed and calculated structure factors, Rfree is calculated for a randomly chosen 5% of reflections, and Rcryst is calculated for the remaining 95% of reflections0.194 Rfree0.219 Protein atoms2,067 Ligand atoms33 Solvent atoms255 Average B-factor40.0 Å2 r.m.s.d.cr.m.s.d., root mean square deviation from target valuesBond lengths0.014 ÅBond angles1.41°a Rsym = ΣIavg — II/ΣIIb Rcryst = ΣFP — FPcalc/ΣFP, where FP and FPcalc are observed and calculated structure factors, Rfree is calculated for a randomly chosen 5% of reflections, and Rcryst is calculated for the remaining 95% of reflectionsc r.m.s.d., root mean square deviation Open table in a new tab Reagents, Plasmids and Mutations for Transcriptional Activity Assays—The COS-7 cells (ATCC, Manassas, VA, CRL1651) and the human osteosarcoma cell line, U-2OS, (ATCC, Crl HTB 96) were purchased from ATCC. Cholesterol and cholesterol sulfate were purchased from Steraloids (Newport, RI). The pCMX-RORα plasmid was a kind gift from Dr. M. Becker-Andre (Serono, Geneva, Switzerland). The reporter ROREtkluc was a kind gift from Dr. V. Giguere (McGill Univ, Montreal, Canada) and has been described previously (12Giguere V. Tini M. Flock G. Ong E. Evans R.M. Otulakowski G. Genes Dev. 1994; 8: 538-553Google Scholar). The RORα mutants C228Q, A330Q,A371Q,C323L, and K339A,E509A were generated using a QuikChange XL site-directed mutagenesis kit from Stratagene (La Jolla, CA). Cell Culture and Transfection Assays—Cells were seeded in 12-well plates at a density of 1 × 105 cells/cm2, 24 h before transfection, and transfected using Fugene6 transfection reagent (Roche Applied Science). A typical reaction mixture contained 0.5 μg pCMX-RORα expression vector, 1.0 μg of reporter plasmid ROREtkluc, 0.1 μg of pCMV-β-galactosidase, and 3 μl of Fugene 6. After a 4-h exposure to the transfection mix, the medium was refreshed and left for another 24 h. Transfected cells were subsequently harvested for the luciferase assay by scraping the cells into 250 μl of passive lysis buffer after washing them in phosphate-buffered saline. Luciferase activity was monitored according to the Promega luciferase assay kit using an automatic luminometer LB96P (Berthold, Regensburg, Germany). Results are expressed in relative light units per β-galactosidase unit. Structure Determination of the Complex RORα LBD-Cholesterol Sulfate—We have expressed human RORα LBD (residues 271–523, numbering according to splice variant 1 of SWISS-PROT entry P35398, and a PreScission™ cleavable N-terminal His tag) in the baculovirus system. After a two-step purification and without cleaving off the N-terminal tag, we exchanged to more than 95% bound cholesterol with cholesterol sulfate (as determined by ESI-MS analysis) and crystallized the complex. Crystals belonging to the monoclinic space group P21 (unit cell: a = 54.4 Å, b = 49.9 Å, c = 60.7 Å, β = 97.8°, 1 complex/asymmetric unit) reached maximal dimensions of up to 0.2 mm in hanging drops at 4 °C within 6 weeks. The structure was solved using the coordinates of the complex of RORα LBD with cholesterol (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). Overall Structure of the Complex RORα LBD-Cholesterol Sulfate—The results of the crystallographic refinement are summarized in Table I. The nomenclature of the secondary structure elements is based on the RXR LBD crystal structure (13Bourguet W. Ruff M. Chambon P. Gronemeyer H. Moras D. Nature. 1995; 375: 377-382Google Scholar) and is identical to the one used for RORα LBD (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). In general, the electron density is of excellent quality, except for amino acids 461–466 (loop L9–10), which had only weak density. The protein part of the refined model consists of the last two His amino acids from the His tag followed by the PreScission™ site (LEVLFQG) and by amino acids 271–511 of the RORα LBD. The refined model also contains 256 water molecules and one cholesterol sulfate molecule. RORα LBD bound to cholesterol sulfate is in an agonist-bound state, as judged by the position of H12 (14Renaud J.P. Moras D. Cell. Mol. Life Sci. 2000; 57: 1748-1769Google Scholar). H12 in this position, together with the H3-H4 region, forms the interaction surface (AF-2) for the coactivator (14Renaud J.P. Moras D. Cell. Mol. Life Sci. 2000; 57: 1748-1769Google Scholar). As found previously for the complex with cholesterol (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar), the PreScission™ cleavage site (LEVLFQGP) acts as a mimic of the LXXLL coactivator sequence and adopts an α-helical conformation. It interacts with the coactivator binding site of a neighboring molecule in the crystal lattice and forms the classical “charge clamp” capping interactions mediated by Lys339(H3) and Glu509(H12). The overall structures of RORα LBD in complex with cholesterol and cholesterol sulfate are very similar. The root mean square deviation using the LSQ option from the program O (10Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar) was 0.26 Å for the 242 Cα atoms from residues Pro270-Phe511. Significant changes in the protein parts of the LBP (Fig. 1A) occur only for the side chain of Ile327 and the loop L1–2 (residues Gln289 and Tyr290). The backbone NH-atoms for Gln289 and Tyr290 move by ca. 0.8 Å toward the sulfate group (with a concomitant movement of the respective side chains), thus improving the interactions with the sulfate group. The side chain of Ile327 has moved slightly, to prevent a steric clash with the terminal isopropyl group of cholesterol sulfate. Cholesterol Sulfate in the LBP of RORα—Cholesterol sulfate is bound in the LBP of RORα. The sulfate group, which is located in the hydrophilic part of the LBP, makes direct hydrogen bond interactions with NH-Gln289 (3.0 Å), NH-Tyr290 (2.9 Å), and a bidentate interaction with NH1-Arg370 (3.0 Å, 3.1 Å). This confirms the docking hypothesis, which had led to the proposal of cholesterol sulfate as a ligand with an improved affinity relative to cholesterol (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). In addition, a water-mediated interaction is made with NH1-Arg367 (Fig. 1A). The comparison shows that cholesterol sulfate and cholesterol have a similar overall mode of binding, but cholesterol sulfate is displaced slightly toward the hydrophilic and positively charged part of the LBP (Fig. 1A). This can be explained by the optimization of electrostatic and hydrogen-bond interactions made by the sulfate group. Interestingly, seven well ordered water molecules present for cholesterol in the hydrophilic part of the LBP have been displaced in the complex with cholesterol sulfate (Fig. 1B). Only one conserved water molecule is still present, which mediates interactions from the sulfate group to NH1-Arg367 and O-Ala330. The average B-value for the ligand (28.7 Å2) is lower than the average B-value for the protein (40.0 Å2), consistent with the fact that excellent electron density for all non-hydrogen atoms of cholesterol sulfate is visible. Cholesterol sulfate adopts thus (like cholesterol) a single, well defined position in the LBP. The following amino acids have a non-hydrogen atom closer than 4 Å to the ligand cholesterol sulfate: Cys288(loop H1-H2), Gln289(loop H1-H2), Tyr290(loop H1-H2), Trp320(H3), Cys323-(H3), Ala324(H3), Lys326(H3), Ile327(H3), Ala330(H3), Val364-(H5), Arg367(H5), Met368(H5), Arg370(H5), Ala371(H5), Val379-(s1), Tyr380(s1), Phe381(s1), Phe391(H6), Leu394(H6), Val403(H7), and His484(H11). Cholesterol Sulfate Is Bound More Tightly than Cholesterol—We used non-denaturing ESI-MS to confirm the exchange of cholesterol by cholesterol sulfate in the RORα LBD. Exchange yield using cholesterol sulfate was greater than 95%. As shown previously (5Bitsch F. Aichholz R. Kallen J. Geisse S. Fournier B. Schlaeppi J-M. Anal. Biochem. 2003; 323: 139-149Google Scholar), the corresponding protein-ligand complex also showed a higher stability to collision-induced dissociation in the electrospray ionization interface. Moreover, the exchange of bound cholesterol sulfate could not be reversed by adding back an excess of cholesterol or other cholesterol derivatives such as hydroxycholesterols (data not shown). In addition, differential scanning calorimetry was used to assess the stabilities of the RORα LDB complexes with cholesterol and cholesterol sulfate, respectively. Fig. 2 shows that cholesterol sulfate dramatically increased the phase transition temperature of RORα LBD by 9 °C, relative to cholesterol. Overall, these results confirm that, as predicted by the x-ray structures, cholesterol sulfate has a higher affinity than cholesterol for RORα LBD. Sulfonation of Cholesterol Improves RORα Transcriptional Activity—To further characterize the possible biological role of cholesterol sulfate for RORα, a series of mutations was designed and evaluated for their effects on the transcriptional activities. The double mutation K339A,E509A (i.e. a “knockout” of the charge clamp) was made as a negative control. Indeed, this double mutant triggered a transcriptional activity similar to the one obtained with an empty vector (Fig. 3A). The triple mutant A330Q,A371Q,C323L (designed to prevent binding of cholesterol and cholesterol sulfate in the LBP) showed a reduced activity (Fig. 3A) consistent with the results obtained previously for the single mutants A330L, A371Q, and C323L (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). A mutation that might prevent binding of cholesterol sulfate more than that of cholesterol was designed based on the crystal structure. We hypothesized that the mutation C288Q would modify the position of Arg370 and/or of loop L1–2 (Fig. 1B) and thus would preferentially lower the affinity of cholesterol sulfate versus cholesterol. We first tested this mutation in cells for which the intracellular cholesterol level was not manipulated. Most likely, as shown in normal epithelial cells, the ratio between cholesterol and cholesterol sulfate in these cells is about 500:1 (15Lin Y.N. Horowitz M.I. Steroids. 1980; 36: 697-708Google Scholar). Under these experimental conditions, the transcriptional activity of the mutant RORα C288Q was found to be similar to the one elicited by RORα wild type (Fig. 3A). This unaffected transcriptional activity of the C288Q mutant probably reflects the binding of cholesterol to the mutated RORα. The intracellular cholesterol level in U2OS cells can be reduced by using lovastatin (hydroxymethylglutaryl-CoA reductase inhibitor) and cyclodextrin, as described previously (3Kallen J. Schlaeppi J-M. Bitsch F. Geisse S. Geiser M. Delhon I. Fournier B. Structure. 2002; 10: 1697-1707Google Scholar). Under these conditions, using RORα wild type, we have shown that cholesterol sulfate elicited an increased transcriptional activity of 160% when compared with cholesterol. In contrast, the mutant C288Q did not display this improved transcriptional activity (with respect to cholesterol) when cells were treated with cholesterol sulfate (Fig. 3B). This is consistent with the prediction that the C288Q mutant preferentially reduces the affinity of cholesterol sulfate. We cannot exclude that the only partial cholesterol depletion in these cells might reduce this observed effect on cholesterol sulfate. It would be interesting to investigate RORα transcriptional activity in cells for which the cholesterol:cholesterol sulfate ratio is more drastically modified such as in keratinocytes during differentiation, where this ratio is as low as 5:1, or in ichtyosis-derived cells, where this ratio is even further reduced to 1:1 (16Epstein Jr., E.H. Krauss R.M. Shackleton C.H. Science. 1981; 214: 659-660Google Scholar, 17Williams M.L. Elias P.M. J. Clin. Invest. 1981; 68: 1404-1410Google Scholar). Recently, there has been an increasing interest in steroid sulfonation, in particular for their potential involvement in breast and prostate cancer (18Strott C.A. Endocr. Rev. 2002; 23: 703-732Google Scholar). It is postulated that estrone sulfate in the breast tumors (19Falany J. Macrina N. Falany C.N. Breast Cancer Res. Treat. 2002; 74: 167-176Google Scholar) could play a role in regulating the level and the activity of 17β-estradiol and that disruption of estrogen sulfotransferase could lead to modulation of the estrogen pathway (20Qian Y.M. Sun X.J. Tong X.P. Li P. Richa J. Song W.C. Endocrinology. 2001; 142: 5342-5350Google Scholar). In our study, we show that sulfonation of cholesterol allows improved binding to RORα, reflected by an increased transcriptional activity. The possible exchange of cholesterol with cholesterol sulfate inside the cells could represent a mode of regulation of intracellular cholesterol level since it has been shown that cholesterol sulfate inhibits cholesterol esterification (21Nagakawa M. Kojima S. J. Biochem. (Tokyo). 1976; 80: 729-733Google Scholar) and that cholesterol sulfate could potently modulate hydroxymethylglutaryl-CoA, the rate-limiting enzyme for cholesterol synthesis (22Williams M.L. Hugues-Fulford M. Elias P.M. Biochim. Biophys. Acta. 1985; 854: 349-357Google Scholar). The identification of RORα target genes possibly involved in these mechanisms could contribute to a better understanding of these regulations by cholesterol sulfate. Despite the fact that cholesterol sulfate is widely distributed in human tissues, its physiological role is not well understood (for a review, see Ref. 23Strott C.A. Higashi Y. J. Lipid Res. 2003; 44: 1268-1278Google Scholar). In skin, an important role for cholesterol sulfate emerged, e.g. in recessive X-linked ichtyosis for which a genetic deficiency in steroid sulfatase leads to the accumulation of cholesterol sulfate in stratum corneum (16Epstein Jr., E.H. Krauss R.M. Shackleton C.H. Science. 1981; 214: 659-660Google Scholar). In addition, the role of cholesterol sulfate in keratinocyte differentiation has been well documented (24Hanley K. Wood L. Ng D.C. He S.S. Lau P. Moser A. Elias P.M. Bikle D.D. Williams M.L. Feingold K.R. J. Lipid Res. 2001; 42: 390-398Google Scholar, 25Jetten A.M. George M.A. Nervi C. Booen L.R. Rearick J.F. J. Investig. Dermatol. 1989; 92: 203-209Abstract Full Text PDF Google Scholar, 26Kawabe S. Ikuta T. Ohba M. Chida K. Ueda K. Yamanishi K. Kuroki T. J. Investig. Dermatol. 1998; 11: 1098-1102Google Scholar, 27Kashiwagi M. Ohba M. Chida K. Kuroki T. J. Biochem. (Tokyo). 2002; 132: 853-857Google Scholar). It would therefore be interesting to reconsider RORα function in organs such as skin and testis, in which cholesterol sulfate was found to be abundant and which display the strongest expression of RORα (28Steinmayr M. Andre E. Conquet F. Rondi-Reig L. Delhaye-Bouchaud N. Auclair N. Daniel H. Crepel F. Mariani J. Sotelo C. Becker-Andre M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3960-3965Google Scholar). In addition, cholesterol sulfate is also a possible precursor of important sulfonated adrenal steroids such as dehydroepiandrosterone sulfate and pregnenolone sulfate (23Strott C.A. Higashi Y. J. Lipid Res. 2003; 44: 1268-1278Google Scholar, 29Mason J.I. Hemsell P.G. Endocrinology. 1982; 111: 208-213Google Scholar). It is worth noting that pregnenolone sulfate, now considered as an essential neurosteroid, is actively synthesized in brain, in particular in Purkinje cells (30Tsutui K. Sakamoto H. Ukena K. J. Steroid Biochem. Mol. Biol. 2003; 85: 311-321Google Scholar). This information could thus also shed new light on the well described cerebellar phenotype of the RORα mutant mice (31Hamilton B.A. Frankel W.N. Kerrebrock A.W. Hawkins T.L. FitzHugh W. Kusumi K. Mueller Russell L K. van Berkel V. Birren B.W Kruglyak L. Lander E.S. Nature. 1996; 379: 736-739Google Scholar). We thank Drs. M. Geiser and S. Geisse for cloning and fermentation and R. Cebe, Y. Pouliquen, A. Berner, and A.Graham for technical assistance. The use of the Novartis modeling software WITNOTP, written by A. Widmer, and the experimental assistance from the Swiss Light Source (PX Beam Line X06SA), C.Schulze-Briese, is acknowledged. We thank H. Widmer, H. P. Kocher, R. Gamse, and M. Missbach for interest and support.