Oxysterols are substrates for cholesterol sulfotransferase

• DOI: 10.1194/jlr.m700018-jlr200
• Publication Date: 2007-03-09T02:21:29Z
• Authors: Hirotoshi Fuda; Normal B. Javitt; Kuniko Mitamura; Shigeo Ikegawa; Charles A. Strott
• Keywords: Oxysterol; Liver X receptor; Viability assay

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation nuclear receptors. The role oxysterols such as 7-ketocholesterol (7-KC) in the development retinal macular degeneration and atheromatous lesions is particular interest, but little known their metabolic fate. We establish steroid/sterol sulfotransferase SULT2B1b, efficiently sulfonate cholesterol, also effectively sulfonates variety oxysterols, including 7-KC. cytotoxic effect 7-KC on 293T cells was attenuated when these cells, which do not express were transfected with SULT2B1b cDNA. Importantly, protection 7-KC-induced loss cell viability transfection correlated synthesis protein production sulfoconjugate (7-KCS). Moreover, 7-KCS added culture medium amounts equimolar 7-KC, no occurred. Additionally, MCF-7 highly significantly more resistant extended range oxysterol substrates for include 7α/7β-hydroxycholesterol 5α,6α/5β,6β-epoxycholesterol well 7α-hydroperoxide derivative cholesterol. Thus, by acting offers potential pathway modulating vivo injurious compounds. 2-hydroxypropyl-β-cyclodextrin Cell Counting Kit-8 electrospray ionization sulfate 3′-phosphoadenosine 5′-phosphosulfate In contrast many studies have established deleterious biologic processes (1.Brown A.J. Mander E.L. Gelissen I.C. Kritharides L. Dean R.T. Jessup W. Cholesterol metabolism subcellular distribution macrophage foam cells. Accumulation oxidized esters lysosomes.J. Lipid Res. 2000; 41: 226-237Abstract Full Text PDF PubMed Google Scholar, 2.Lemaire-Ewing S. Prunet C. Montange T. Vejux A. Berthier Bessede G. Corcos Gambert P. Neel D. Lizard Comparison cytotoxic, pro-oxidant pro-inflammatory characteristics different oxysterols.Cell Biol. Toxicol. 2005; 21: 97-114Crossref Scopus (168) 3.Leonarduzzi Vizio B. Sottero Verde V. Gamba Mascia Chiarpotto E. Poli Biasi F. Early involvement ROS overproduction apoptosis induced 7-ketocholesterol.Antioxid. Redox Signal. 2006; 8: 375-380Crossref (63) 4.Smith L.L. Johnson B.H. Biological activities oxysterols.Free Radic. Med. 1989; 7: 285-332Crossref (333) Scholar), there been few focusing pathways disposal. Of interest are high levels atheromas because association instability rupture, prelude myocardial infarction (5.Carpenter K.L. Taylor S.E. van der Veen Williamson B.K. Ballantine J.A. Mitchinson M.J. Lipids oxidised lipids human atherosclerotic at stages development.Biochim. Biophys. Acta. 1995; 1256: 141-150Crossref (178) 6.Garcia-Cruset Carpenter Guardiola cap core advanced lesions.Free 1999; 30: 341-350Crossref (51) Scholar). A major found other tissues (7-KC), induce injury concentrations present (7.Leonarduzzi Kadl Robbesyn Calogero R.A. Leitinger N. Sevanian et al.Oxysterol-induced up-regulation MCP-1 expression cells.Free 39: 1152-1161Crossref (72) 8.Lizard Monier Cordelet Gesquiere Deckert Gueldry Largrost Characterization comparison mode death, versus necrosis, 7beta-hydroxycholesterol vascular wall.Arterioscler. Thromb. Vasc. 19: 1190-1200Crossref (186) Scholar); this reason, exists focus can lead reduction its toxicity. For example, it has shown substrate 27-hydroxylation, thus forming water-soluble triol decreases intracellular concentration macrophages (9.Babiker Andersson O. Lund Xiu R.J. Deeb Reshef Leitersdorf Diczfalusy U. Bjorkhem I. Elimination endothelial sterol 27-hydroxylase mechanism. density lipoprotein-mediated reverse transport.J. Chem. 1997; 272: 26253-26261Abstract (204) 10.Brown Watts G.F. Burnett J.R. Sterol acts culture.J. 275: 27627-27633Abstract (71) 11.Jessup Brown Novel routes 7-ketocholesterol.Rejuvenation 9-12Crossref (33) recently reported addition accelerating transport, 27-hydroxylation prevents coculture nullifies toxicity (12.Lee J.W. Fuda H. Javitt N.B. Strott C.A. Rodriguez I.R. Expression localization (CYP27A1) monkey retina.Exp. Eye 83: 465-469Crossref (49) findings consonant differential used combination (13.O'Sullivan O'Callaghan Y.C. O'Brien N.M. Different mixtures oxidation products bovine aortic monocytic U937 cells.Int. J. 24: 173-179Crossref (18) Another became apparent one member SULT2 family cytosolic sulfotransferases, affinity (14.Javitt Lee Shimizu hydroxycholesterol sulfotransferases: identification, distinction dehydroepiandrosterone sulfotransferase, tissue expression.Endocrinology. 2001; 142: 2978-2984Crossref (75) Although recognized sulfonation steroid hormones affects activity (15.Hähnel R. Twaddle Ratajczak specificity estrogen receptor uterus.J. Steroid Biochem. 1973; 4: 21-31Crossref (125) Scholar) influence disposal (16.Bongiovanni A.M. Cohn R.M. Clinical aspects conjugation.in: Bernstein Solomon Chemical Aspects Conjugation. Springer-Verlag, New York1970: 409-453Crossref concept an analogous C27 sterols received limited attention. Cytosolic sulfotransferases make up superfamily enzymes steroids/sterols (17.Nagata K. Yamazoe Y. Pharmacogenetics sulfotransferase.Annu. Rev. Pharmacol. 40: 159-176Crossref (177) further differentiated into two subfamilies: SULT2A1, prototypical commonly referred SULT2B1. SULT2B1 gene, alternative exon 1 splicing, encodes isoforms, SULT2B1a (18.Her Wood T.C. Eichler E.E. Mohrenweiser H.W. Ramagli L.S. Siciliano Weinshilboum Human hydroxysteroid SULT2B1: encoded single chromosome 19 gene.Genomics. 1998; 53: 284-295Crossref (135) Whereas SULT2A1 avidly pregnenolone, they use substrate; hand, isozyme highest efficiency and, therefore, represents physiologic (19.Fuda Mutational analysis isoforms reveals 1B gene produces whereas 1A yields pregnenolone sulfotransferase.J. 2002; 277: 36161-36166Abstract (84) report, we evidence than isozyme. Interestingly, however, using able level additional studies, 5α,6α/5β,6β-epoxy, 7α/7β-hydroxy, expanding novel both hydroperoxides. Finally, first time does indeed occur vivo, demonstrated tissue. Cholesterol, 7α-/7β-hydroxycholesterol, 5α,6α-/5β,6β-epoxycholesterol purchased Steraloids (Newport, RI). Methanol, acetonitrile, ammonium acetate liquid chromatography-mass spectrometry HPLC grade obtained Nacalai Tesque (Kyoto, Japan). Distilled water Wako Pure Industries (Osaka, prepared hematoporphyrin visible light according procedure described detail previously (20.Rodriguez-Estrada M.T. Costa Pedillo M. Caboni M.F. Lercker product preparation methods subsequent gas chromatographic analysis.J. AOAC Int. 2004; 87: 474-480Crossref isotopes [35S]3′-phosphoadenosine (PAPS; 1.1 Ci/mmol) [3H]7-KC (40 Perkin-Elmer Life Sciences (Boston, MA) American Radiolabeled Chemicals (St. Louis, MO), respectively. Iodine crystals, (BCD), PAPS, polyethylenimine Sigma-Aldrich MO). Organic solvents either Baker or Mallinckrodt (Phillipsburg, NJ). Silica gel TLC plates Analtech (Newark, DE), Immobilon-P Millipore (Bedford, MA). TOPO TA Cloning Kit, pcDNA3.1(+), DMEM, FBS, Antibiotic-Antimycotic Invitrogen (Carlsbad, CA). PfuUltra Hotstart DNA Polymerase Stratagene (La Jolla, Oligonucleotides Operon Biotechnologies (Huntsville, AL). (CCK-8) Dojindo Molecular Technologies (Gaithersburg, MD). Goat anti-rabbit IgG conjugated horseradish peroxidase LumiGLO Chemiluminescent Substrate System KPL Proteinase inhibitor cocktail Roche (Indianapolis, IN), BCA assay kit Pierce (Rockford, IL). BioMax XAR film Kodak (Rochester, NY). Melting point measured micro hot-stage apparatus uncorrected. 1H-NMR spectrum recorded JEOL EX-270 spectrometer (JEOL, Tokyo, Japan) operated 270.05 MHz. shifts given δ value tetramethylsilane internal standard (s, singlet; d, doublet; m, multiplet). LC-MS performed Finnigan LTQ linear ion-trap mass (Thermo Electron, San Jose, CA) equipped (ESI) source coupled Paradigm MS4 pump (Michrom Bioresources, Auburn, autosampler (HTC PAL; CTC Analytics, Zwingen, Switzerland). conditions verifying structure (7-KCS) follows: ion voltage, −4 kV; capillary temperature, 270°C; −33 V; sheath (nitrogen gas) flow rate, 50 arbitrary units; auxiliary 5 tube lens offset −135 tandem MS analysis, helium collision normalized energy set 25%. LC separations conducted semimicro column, TSKgel ODS-100V (5 μl, 150 × 2 mm inner diameter; Tosoh Co., isocratic elution acetonitrile-5 mM buffer, pH 6.0 (3:1, v/v), mobile phase rate 200 μl/min. To prepare sodium salt 7-KCS, solution consisting mg ml dry pyridine freshly sulfur trioxide-pyridine complex stirred room temperature overnight. After evaporation vacuo residue redissolved passed through short pad Cosmosil 140C18-OPN (Nacalai Tesque) sintered glass filter. washing water, eluted methanol eluate chromatographed silica chloroform-methanol (7:1, v/v). yield 100%, colorless solid had melting 125–129°C. ESI-MS, m/z 479.4 [M-H]− (100%); 96.8 [HSO4]−. (CDCl3) δ: 0.683 (3H, s, H-18), 0.863 J = 6.48 Hz, H-26 H-27), 0.867 6.75 0.923 H-21), 1.197 H-19), 4.338 (1H, H-3α), 5.700 H-6). SULT2B1a, overexpressed bacteria glutathione S-transferase fusion proteins, cleaved, affinity-purified 20 μl reaction volume contained 0.1 PAPS purified enzyme Briefly, mixture (4 μg), (1 μg) Tris-HCl buffer (pH 7.5) containing MgCl2, 0.2 BCD, 4% ethanol prepared. Reactions carried out 37°C min stopped 100°C min. adding 10 mg/ml carrier, aliquots applied plates. Chromatography solvent system chloroform-methanol-acetone-acetic acid-water (8:2:4:2:1), after dried exposed iodine vapor visualize location 7-KCS. iodine-adsorbed spots excised placed counting vials scintillation cocktail, radioactivity determined spectrometry. above. 7.5), substrate, μM [35S]PAPS. amount each (μg/tube) (0.4 7α-hydroxycholesterol 7β-hydroxycholesterol 5α,6α-epoxycholesterol (0.1 5β,6β-epoxycholesterol μg). Enzymatic reactions, chromatography, staining, PCR isolate pcDNA-SULT2B1b. amplified pGEX-6P-3-SULT2B1b template SULT2B1b-specific primers 5′-TCTAGAATGGACGGGCCCGCCGAGCCCCAGATC-3′ (sense) 5′-GCGGCCGCTTATGAGGGTCGTGGGTG-3′ (antisense). underlined areas indicate XbaI NotI sites, denaturing 95°C min, followed 25 cycles 30 annealing 55°C extension 72°C subcloned pCR2.1-TOPO Kit manufacturer's protocol sequenced. digestion NotI, ligated XbaI/NotI-digested pcDNA3-cMyc (kindly provided Dr. Inohara Naohiro, Department Pathology, University Michigan Medical School). pcDNA-PAPS synthetase (PAPSS1), pGEX-6P-3-PAPSS1 BamHI BamHI/NotI-digested pcDNA3.1(+) (21.Fuda Akita bifunctional synthase isoforms.Biochem. 364: 497-504Crossref generously Pamela Schwartzberg National Genome Research Institute (National Institutes Health, Bethesda, Type Culture Collection (Manassas, VA). Cells grown DMEM supplemented 10% (v/v) FBS 5% CO2. Media changed every day. seeded 6 cm Falcon dishes (Franklin Lakes, NJ) 16.5 105 cells/dish day before being transfected. Transfections Calphos Mammalian Transfection BD Biosciences Clontech (Mountain View, pcDNA-SULT2B1b plus pcDNA-PAPSS1 empty vector control mixed M CaCl2 HEPES-buffered saline incubated mixture, media 8 h Transfected (48 h) (10 104) without phenol red 24-well coated 0.05% cytotoxicity, (Dojindo Technologies). Either dissolved 45% (w/v) BCD media, CO2 24 h. period, CCK-8 cultures incubations continued 1.5 1% dodecyl solution. Care taken avoid exposure. Absorbance 450 nm μQuant plate reader (BioTek, Winooski, VT). Data statistically analyzed two-way ANOVA GraphPad Prism (San Diego, pcDNA3.1 48 then reseeded dish cells/dish. [3H]7-KCS nM) delipidated (Monobind, Lake Forest, extracted above formation. model 1100 Hewlett-Packard instrument photoarray detector 210 225 nm. C18 reverse-phase 250 4.6 chromatograph 4 μ column (catalog number OOG-4375-EO; Phenomenex, Torrance, binary methanol-water beginning 30% increasing 100% over period continuing isocratically constant 0.8 ml/min. Samples counted (Beckman). washed times PBS disrupted plastic scraper cold RIPA lysis (20 Tris-HCl, 7.5, NaCl, 0.1% SDS, deoxycholate, Triton X-100) proteinase cocktail. centrifugation supernatant collected kit. subjected electrophoresis NuPage 4–12% Bis-Tris (Invitrogen) MOPS/SDS running transferred polyvinylidene fluoride membrane (Immobilon-P). Membranes soaked milk TBS Tween (T-TBS) gentle shaking. (1:2,000; HL4360 #9) PAPSS1 HL4004) overnight 4°C three T-TBS (1:55,000) temperature. chemiluminescent exposing membranes X-ray previously. quantifying number, chloroform/1% X-100 organic collected. chloroform removed heat block Savant SpeedVac (GMI, Ramsey, MN). content Cholesterol/Cholesteryl Ester Quantitation instructions (BioVision, Mountain expressed μg/106 Fresh-frozen specimens atheroma, autopsies under approved protocol, supplied Allen Burke CVPath Approximately 500 sections 9.5 methanol, microtip probe (Sonicator 3000; Misonix, Farmingdale, NY), samples sonicated (2.5 W) intervals total 3 monitoring cooling ice so did increase >60°C. centrifuged, decanted dryness. 0.5 analysis. Reverse-phase (Aqua μ, 125 A; Phenomenex) maintained 60°C. gradient increased 40 Under conditions, standards retention 26.5 34 respectively, multi-wavelength (210, 225, 233 nm). Methanolic atheroma injected onto fractions Fractions 26 28 pooled specimen lyophilized 75% LC-ESI-MS That penchant sterols, isozymes, Fig. . order magnitude active sulfonating (Table 1). ability α- β-isomers 7-hydroxycholesterol 5,6-epoxycholesterol 2). although sulfonated efficiency, nearly good 7α-hydroxycholesterol, 3). By contrast, formation <10% occurring (data shown).TABLE 1.SULT2 kinetic valuesEnzymeKmVmaxkcatkcat/KmMnmol/min/mgs−1M−1 s−1SULT2B1b7.7 10−629.12.0 10−22.6 103SULT2B1a2.4 10−56.74.2 10−31.8 102SULT2A11.7 10−57.03.9 10−32.3 1027-KC, 7-ketocholesterol. affinity-purified. (PAPS), [3H]7-KC. reactants Experimental Procedures. Open table new tab TABLE 2.SULT2B1b valuesSubstrateKmVmaxkcatkcat/KmMnmol/min/mgs−1M−1 s−1Cholesterol1.1 10−625.21.7 10−21.6 1047α-Hydroxycholesterol2.3 10−650.33.5 10−21.5 1045α,6α-Epoxycholesterol2.5 10−619.71.4 10−25.4 1037-KC5.5 10−638.22.6 10−24.8 1037β-Hydroxycholesterol5.9 10−636.22.5 10−24.2 1035β,6β-Epoxycholesterol3.9 10−622.61.6 10−24.0 103The μl) consisted optimal protein, affinity-purified, 3.SULT2B1b oxysterolsSterol SubstrateEnzyme Activitynmol/min/mgCholesterol7.57-KC7.17α-Hydroxycholesterol21.07β-Hydroxycholesterol1.97α-Hydroperoxide4.8Reactions 0.08 Tris 7.1) 87 [35S]PAPS, 1.3 BCD. g/ml, [35S]sterol extraction methylene blue