{beta}-Apocarotenoids do not significantly activate retinoic acid receptors {alpha} or {beta}

Abstract

beta-Carotene oxygenase 2 cleaves beta-carotene asymmetrically at non-central double bonds of the polyene chain, yielding apocarotenal molecules. The hypothesis tested was that apocarotenoids are able to stimulate transcription by activating retinoic acid receptors (RARs). The effects of long- and short-chain apocarotenals and apocarotenoic acids on the activation of RARalpha and RARbeta transfected into monkey kidney fibroblast cells (CV-1) were investigated. We synthesized or purified beta-apo-8'-carotenoic acid (apo-8'-CA), beta-apo-14'-carotenoic acid (apo-14'-CA), beta-cyclocitral (BCL), beta-cyclogernanic acid (BCA), beta-ionone (BI), beta-ionylideneacetaldehyde (BIA) beta-ionylideneacetic acid (BIAA) and a C13 ketone, beta-apo-13-carotenone (C13). None of the apocarotenoids tested showed significant transactivation activity for the RARs when compared with all-trans retinoic acid (RA). The results suggest that biological effects of these apocarotenoids are through mechanisms other than activation of RARalpha and beta.

Figure 1

Metabolism of β-carotene. The products formed when cleaved centrally by β,β-carotene 15,15′-oxygenase (BCO1) and eccentrically by β-carotene 9′10′-oxygenase (BCO2). BCO1 catalyzes the main pathway of β-carotene metabolism, which cleaves the central 15,15′-double bond of β-carotene to retinal. Retinal as a vitamin A precursor is converted to retinol or further oxidized to retinoic acid in vivo. In comparison, BCO2 catalyzes the eccentric cleavage which cleaves at the double bonds other than the central 15, 15′-double bond of the polyene chain of β-carotene. β-Apocarotenals may be further oxidized to β-apocarotenoic acids

Figure 2

Possible metabolites of β-carotene when eccentrically cleaved. Boxed compounds were tested to see if they could transactivate retinoic acid receptor α or β

Figure 3

The transactivation of RARE-luciferase receptor in monkey kidney fibroblast cells transfected with retinoic acid receptor (RAR)α treated with varying concentrations of retinoic acid

Figure 4

Retinoic acid receptor (RAR)α transactivation response to retinoic acid (RA) and apocarotenoids in monkey kidney fibroblasts. Cells transfected with RARα receptor vector were treated for 22–24 h with an apocarotenoid concentration of 10⁻⁵ mol/L; the bars from left to right indicate the effects on RARα of RA, apo-14′-CA, apo-8′-CA, β-ionone, β-cyclocitral, β-ionylideneacetic acid and C13 ketone, respectively. At concentrations of 10⁻⁶ mol/L, the effects on RARα of RA, apo-8′-CA, apo-14′-CA and C13 ketone were shown by the bars from left to right, respectively. Luciferase activities were measured by dual-luciferase reporter system and were expressed as fold induction in treated cells (see Materials and methods). Data are expressed as the mean ± SD or range from multiple experiments as indicated (n) with three independent observations per experiment. If no ‘n’ is indicated the experiment was conducted once in triplicate

Figure 5

Response of retinoic acid receptor (RAR)β transactivation by retinoic acid (RA) and β-carotene metabolites in monkey kidney fibroblast. Cells transfected with RARβ receptor vector were treated for 22–24 h with a concentration of 10⁻⁵ mol/L, the bars from left to right indicate the effects on RARβ of RA, apo-14′-CA, apo-8′-CA, β-ionone, β-cyclocitral, β-ionylideneacetic acid, C13 ketone, β-ionylideneacetaldehyde and β-cyclogeranic acid, respectively. At concentrations of 10⁻⁶ mol/L, the effects on RARβ of RA, apo-8′-CA, apo-14′-CA and C13 ketone were shown by the bars from left to right, respectively. Luciferase activities were measured by dual-luciferase reporter system and were expressed as fold induction in treated cells (see Materials and methods). Data are expressed as the mean ± SD or range from multiple experiments as indicated (n) with three independent observations per experiment. If no ‘n’ is indicated the experiment was conducted once in triplicate