Naturally occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors

Abstract

β-Carotene is the major dietary source of provitamin A. Central cleavage of β-carotene catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA), which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of β-carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β-apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs. Importantly, however, β-apo-14'-carotenal, β-apo-14'-carotenoic acid, and β-apo-13-carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can interact directly with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14'-carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nm β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β-carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

FIGURE 1.

β-Apocarotenoids. Structures of the β-apocaroteniods were synthesized (indicated by [S]), purified, and characterized for this study. r = CHO in the carotenals and r = COOH in the carotenoic acids.

FIGURE 2.

Chemical synthesis of all possible β-apocarotenoids. Reagents and conditions used in the synthesis of the various compounds are shown in lowercase roman numerals, and yields are shown in parentheses. i, 5 n KOH/EtOH (± benzene); room temperature, 12 h (99%, β-ionone; 96%, β-apo-12′-carotenoic acid; 92%, β-apo-10′-carotenoic acid; 94%, β-apo-14′-carotenoic acid). ii, LiAIH₄, tetrahydrofuran, room temperature, 45 min (for compound 2, 9); DIBAL-H, CH₂Cl₂, room temperature, 30 min (for compound 6). iii, MnO₂, Celite, CH₂Cl₂, room temperature, 4 h (73%, β-ionylideneacetaldehyde; 36%, β-apo-10′-carotenal; 6%, β-apo-14′-carotenal). iv, (triphenylphosphoranylidene)-2-propanone, toluene, reflux, 12 h (61%). v, NaH, dialdehyde shown, CH₂Cl₂, 0 °C to room temperature, 48 h (59%). vi, KCN, CH₃COOH, MnO₂, MeOH, room temperature, 90 h (21%, 4; 2%; 10). vii, NaH, triethylphosphonoacetate, THF, 0 °C to room temperature, 48 h (83%, 5; 94%, 1; 74%, 8). viii, O₂, CH₂Cl₂, 48 h (quantitative).

FIGURE 3.

β-Apocarotenoids do not transactivate retinoic acid receptors. Histograms of activation of RARE reporter genes in cells transfected with retinoic acid receptors α (left), β (middle), and γ (right). Normalized fold activation relative to vehicle-treated cells is shown for all-trans-retinoic acid (far left bar in each histogram) or the β-apocarotenoids resulting from cleavage at the “a”, “b”, “c”, or “d” sites from top to bottom, respectively. Compounds were tested individually at 10⁻⁵ m (n = 3–6); mean ± S.D. Compound definitions are given on Fig. 1.

FIGURE 4.

β-Apocarotenoids antagonize ATRA-induced transactivation of retinoic acid receptors. Histograms of activation of RARE reporter genes in cells transfected with retinoic acid receptors α (left), β (middle), and γ (right). Percent of maximal activation of cells treated with 10⁻⁵ m ATRA alone (left most bar in each histogram) or co-treated with 10⁻⁵ m ATRA and 10⁻⁵ m of the β-apocarotenoids resulting from cleavage at the a, b, c, or d sites are shown in a, b, c, and d, respectively (n = 3 to 6); mean ± S.D. Compound abbreviations are given on Fig. 1.

FIGURE 5.

β-Apo-13-carotenone is a potent antagonist of retinoic acid receptor-mediated induction of reporter gene expression and blocks ATRA induction of endogenous gene expression. a, dose response curves for transactivation of RARγ (left upper panel) by ATRA in the absence (filled diamonds) or presence of 1 nm (green filled triangles), 10 nm (×), or 100 nm (blue filled triangles) C13 ketone. Points shown are the means of six determinations for ATRA alone or three determinations for each of the curves with C13 ketone. Variations about the means were generally <10% except at very low concentrations of ATRA. b, induction of expression of mRNAs for RARβ (left lower panel) or cytochrome P450, 26A1 (CYP26A1) (right lower panel) by 10 nm ATRA treatment alone or by co-treatment with ATRA and the test compounds at 10 nm, including β-carotene (BC), β-ionylideneacetic acid (BIAA), β-apo-14′-carotenal (14′-AL), β-apo-14′-carotenoic acid (14′-CA), and β-apo-13-carotenone (C13 ketone). mRNA levels were quantified by RT-PCR and are shown as the fold induction compared with vehicle-treated cells (n = 3); mean ± S.D.

FIGURE 6.

β-Apo-13-Carotenone is a high affinity ligand for purified retinoic acid receptors and fits into the ligand binding site. a, competitive displacement of 5 nm tritiated ATRA from purified RAR proteins by unlabeled ATRA(♦)as a positive control, C13 ketone (▴), 14′-CA (+), 14′-AL (×), and 13-cis-retinoic acid (■) as a negative control for RARα (left) experiment, CD 2665 (●), retinyl acetate (■) as a negative control for RARβ (middle) and RARγ (right) experiments. Points shown are means of n = 3 with a variance of <10%. b, binding affinities (in nm) of β-apocarotenoids to RARs calculated from the data shown in a, and additional experiments with β-apo-12′- and β-apo-10′-carotenoic acids. For ATRA and the C13 ketone variance shown is for three independent experiments. c, molecular modeling of the docking of ATRA (red) and β-apo-13-carotenone (purple) into the ligand binding site (protein backbone in green) of RARβ (Protein Data Bank code 1XAP) (left). On the right is shown the energy minimized then docked conformations of ATRA (red) and β-apo-13-carotenone (purple) overlaid onto the conformation of the agonist tetramethyl tetrahydronaphthalenyl propenyl benzoic acid (TTNPB) (white) as observed in the x-ray structure.

FIGURE 7.

β-Apo-13-carotenone is a high affinity ligand for purified retinoid X receptor α. a, competitive displacement of 10 nm tritiated 9-cis-RA from purified RXRα protein by unlabeled 9-cis-RA(♦)as a positive control, C13 ketone (▴), 14′-AL (■), 14′-CA (+), and retinyl acetate (×) as a negative control. Points shown are means of n = 3 with a variance of <10%. b, binding affinities of β-apocarotenoids to RXRα calculated from the data shown in a.

FIGURE 8.

Analysis of β-apo-13-carotenone in human plasma by HPLC/MS. Multiple reaction monitoring chromatogram of β-apo-13-carotenone in blood plasma (top) and a standard (bottom) as analyzed by atmospheric pressure chemical ionization in positive mode after C30 HPLC. The multiple reaction monitoring was composed of three transitions m/z 259.2 > 175.1 (blue), 119.1 (red), and 69.0 (green) and the matching elution time and relative intensities of the transitions confirm the peak identity.