A ligand for the aryl hydrocarbon receptor isolated from lung Jiasheng Song†, Margaret Clagett-Dame†‡, Richard E. Peterson‡, Mark E. Hahn§, William M. Westler†, Rafal R. Sicinski†¶, and Hector F. DeLuca†ʈ †Department of Biochemistry, College of Agricultural and Life Sciences, and ‡Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, Madison, WI 53706; and §Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 Communicated by Henry Lardy, University of Wisconsin, Madison, WI, September 17, 2002 (received for review August 20, 2002) The aryl hydrocarbon receptor (AHR) is a ligand-inducible tran- methyl ester (ITE) by UV and IR spectrophotometry, MS, scription factor that is best known because it mediates the actions extensive NMR studies, and quantum mechanical calculations. of polycyclic and halogenated aromatic hydrocarbon environmen- tal toxicants such as 3-methylcholanthrene and 2,3,7,8-tetrachlo- Materials and Methods rodibenzo-p-dioxin. We report here the successful identification of UV Spectroscopy. About 3 ␮g of the ligand in 200 ␮l of methanol͞ an endogenous ligand for this receptor; Ϸ20 ␮g was isolated in water (60:40, vol͞vol) was injected into a cyano HPLC column pure form from 35 kg of porcine lung. Its structure was deduced as (Adsorbosphere CN-AQ 5U, i.d. ϭ 4.6 mm, Alltech Associates) 1؅H-indole-3؅-carbonyl)-thiazole-4-carboxylic acid methyl ester equilibrated with 60% methanol in water at a flow rate of 0.5)2- from extensive physical measurements and quantum mechanical ml͞min. Compounds eluted from the column passed through the calculations. In a reporter gene assay, this ligand activates the AHR flow cell (path length, 10 mm; i.d., 0.009 inches) of a Waters 996 with a potency five times greater than that of ␤-naphthoflavone, photodiode array detector. The UV absorbance of the mobile a prototypical synthetic AHR ligand. 2-(1؅H-indole-3؅-carbonyl)- phase was automatically set to zero. UV absorption spectra were thiazole-4-carboxylic acid methyl ester competes with 2,3,7,8- recorded with MILLENNIUM 3.2 software (Waters) at a rate of one [3H]tetrachlorodibenzo-p-dioxin for binding to human, murine, spectrum per s. The spectrum acquired at the moment of the and fish AHRs, thus showing that AHR activation is caused by direct highest UV absorption is shown. receptor binding, and that recognition of this endogenous ligand is conserved from early vertebrates (fish) to humans. Electron Impact MS. A new glass probe was inserted into a MS50TC Ultrahigh Resolution Mass Spectrometer (Kratos An- he Ah locus was first defined in inbred mice as a strain alytical Instruments), with electron energy of 70 eV and ion Tdifference in response to treatment with polycyclic aromatic source temperature of 150°C. The instrument and probe were at hydrocarbons such as 3-methylcholanthrene (1). The evidence first operated in this mode until a satisfactory background was for the presence of a receptor encoded by the locus was then achieved. A blank spectrum of the residue of column effluent provided by characterizing the strain-specific and stereo-specific eluting at the retention time of the putative ligand was recorded. Ϸ binding sites in mouse liver cytoplasm (2). The coding sequence The sample ( 200 ng) was then introduced on the same probe for the receptor, a soluble protein named aryl hydrocarbon and the spectrum was taken. Any signal common to both the receptor (AHR), was eventually identified and characterized (3). blank and the sample spectrum was subtracted generating the The receptor is found in many vertebrates including fish. The spectrum reported. A KRATOS DS-55 data acquisition system was AHR is a ligand-inducible transcription factor, a member of a used to record all spectroscopic data. basic helix–loop–helix͞Per-Arnt-Sim (bHLH͞PAS) family. On binding to xenobiotic ligands, the AHR has been shown to Fourier Transform (FT)-IR Spectroscopy. One microgram of the regulate the expression of a variety of genes including those ligand in methanol was spotted onto a Teflon STI IR Card encoding for cytochrome P450 enzymes. In addition, receptor (Thermo Spectra-Tech, Shelton, CT). The sample was applied to activation has been linked to alterations in cell proliferation, the card in such a way that the area through which the smallest apoptosis, adipose differentiation, tumor promotion, immune diameter IR beam passes was kept at a minimum to maximize the function, vitamin A status, development, and reproductive func- sample path length with limited supply of material. The solvent tions (4–9). The generation of AHR-deficient mice also points was evaporated and the card was placed in the sample chamber to possible physiological functions of the receptor in liver, heart, of an Infinity 60 AR FT-IR Spectrometer (Thermo-Mattson, ovary, and vascular and immune systems (7, 10–12). Madison, WI). The spectrum was acquired with WINFIRST 3.00 The AHR is best known because polycyclic aromatic hydro- software (resolution, 2; scan number, 200; Iris opening, 1%; carbons, such as 3-methylcholanthrene and benzo[␣]pyrene, and window, KBr). A blank spectrum was generated by using the halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlo- residue from the HPLC effluent as described for MS. rodibenzo-p-dioxin (TCDD), serve as ligands (1, 13). Although the ability of AHR to bind a variety of xenobiotic ligands is of One-Dimensional NMR Spectroscopy. Two micrograms of the ligand great interest, it is clear that the AHR did not evolve to respond in specified solvents was placed in either a 2.5-mm NMR sample to manufactured chemicals. It is reasonable to suspect that tube (Wilmad–Labglass, Buena, NY) or a 5-mm Shigemi tube endogenous ligands must exist for the AHR. Recently, two (Shigemi, Allison Park, PA). The magnetic susceptibility of the Shigemi tube was adjusted to that of the solvent. The standard human urinary products were isolated that bind to the AHR (14). 1 Whether those products are physiological AHR ligands or not is H NMR, single-frequency decoupling, and single frequency undetermined because the identified compounds are indigo, a commonly used fabric dye, and indirubin, an isomer of indigo. Abbreviations: AHR, aryl hydrocarbon receptor; TCDD, 2,3,7,8-tetrachlorodibenzo-p- For the past few years we have labored under the assumption that dioxin; ITE, 2-(1ЈH-indole-3Ј-carbonyl)-thiazole-4-carboxylic acid methyl ester; BNF, endogenous ligands exist for the AHR in tissues of higher ␤-naphthoflavone; HSQC, heteronuclear single quantum correlation; HMBC, hetero- vertebrates. We have now succeeded in isolating from porcine nuclear multiple bond correlation. lung tissue a ligand for AHR and have unequivocally identified ¶Present address: Department of Chemistry, University of Warsaw, Warsaw, Poland 02-093. it as 2-(1ЈH-indole-3Ј-carbonyl)-thiazole-4-carboxylic acid ʈTo whom correspondence should be addressed: E-mail: [email protected]. 14694–14699 ͉ PNAS ͉ November 12, 2002 ͉ vol. 99 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.232562899 Downloaded by guest on September 26, 2021 nuclear Overhauser effect difference spectra were obtained on ppm for 13C (18). The theoretical proton and carbon shifts of Bruker DMX-750 and DMX-500 spectrometers (Bruker, Bil- tetramethysilane were set to zero. lerica, MA). The spectrum of a blank produced as described for Coupling constants were calculated by the single-finite- MS was also recorded. perturbation method (19). The magnitude of the perturbation was 0.02 atomic units, which is within the linear region of Two-Dimensional NMR Spectroscopy. Twelve micrograms of the response, and the calculation was performed at the B3LYP͞6– ligand in deuterated methanol was placed in a 5-mm Shigemi 311ϩg* level of theory. In this method, only the Fermi contact tube (Shigemi). The two-dimensional 1H{13C} heteronuclear contribution to the scalar coupling is available. single quantum correlation (HSQC) and heteronuclear multiple- bond correlation (HMBC) experiments were performed on the Identities of Protons and Carbons in Structure Analysis. For the DMX-500 equipped with a 5-mm triple-resonance, single-axis convenience of identification of atoms in the compound, protons gradient Cryoprobe (Bruker). In the case of the HMBC exper- and carbons are named after their chemical shifts. For example, iment, the direct peaks were not suppressed and a refocusing a proton with a chemical shift of 9.25 ppm will be named as 9.25 180° pulse was applied to both 1H and 13C resonances at the proton and, similarly, a carbon of 141.05 ppm as 141 carbon. midpoint of the antiphase buildup period to refocus static field inhomogeneities. Cell Culture and Reporter Gene Assay. The cell line H1L1.1C2 (20) was maintained in a culture medium (DMEM ϩ 10% FBS) with ␮ ͞ Quantum Mechanical Calculations. Proposed structures were con- G418 at a concentration of 400 g ml and incubated at 37°Cin 6% CO . A 96-well, sterile cell culture plate with about 30,000 structed by using the GAUSSVIEW program (Gaussian, Carnegie, 2 cells per well in 200 ␮l of culture medium without G418 was PA). These models were subjected to full, unconstrained geom- prepared and incubated for at least 12 h before dosing with AHR etry optimization at the B3LYP͞6–31g* level of theory by using ligands in DMSO (0.5% in culture medium) for the reporter the JAGUAR quantum chemical program (Schrodinger, Portland, gene assay. The cells were incubated for an additional4hafter OR). The absence of imaginary frequencies assured that the dosing. The cells were washed twice with PBS (pH 6.9) and lysed models were in a local ground state. Calculated frequencies were by adding 50 ␮l of Cell Culture Lysis Reagent (Promega). Lysate scaled by using the Modified Scaled Quantum Mechanical Force from each well (10 ␮l) was transferred into a corresponding well Fields (SQM) method on the optimized structures (15).