Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: the role of a gene enhancer in the liver chromatin

Abstract

The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D(3)-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity [Gachon, F, Olela, FF, Schaad, O, Descombes, P and Schibler, U, 2006. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. 4, 25-36.; McElwee, JJ, Schuster, E, Blanc, E, Thomas, JH and Gems, D, 2004. Shared Transcriptional Signature in Caenorhabditis elegans Dauer Larvae and Long-lived daf-2 Mutants Implicates Detoxification System in Longevity Assurance. J. Biol. Chem., 279, 44533-43.]. Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month- and 20-month-old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D(3) and the catatoxic synthetic steroid PCN (pregnenolone-16alpha-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-alpha (retinoid X receptor) and PXR in mice injected with D(3) or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.

Figure 1. Sult2A1 gene induction in the liver of vitamin D₃-injected mice

1A) Induction of Sult2A1 mRNAs as assayed by semi-quantitative RT-PCR. 4-month-old mice were injected either with vehicle (0.05M propylene glycol) or D_3, using two mice in each treatment group. 1B) Activity of the IR0 enhancer to recruit VDR, RXR-α, RNA polymerase II (pol II), SRC-1, SRC-2 and SRC-3 in the D₃-injected mice. In tissue ChIP assay, IR0-containing liver chromatin fragments of the mouse Sult2A1 promoter were immunoprecipitated with the test antibodies and the specifically immunoprecipitated DNAs were analyzed on an agarose gel subsequent to PCR amplification. The COX-2 antibody served as a negative control.

Figure 2. Vitamin D₃ responsiveness of the Sult2A1 gene in young and old mice

2A) RT-PCR analysis of Sult2A1 mRNAs in the livers of vitamin D₃ –injected 4 month and 20 month old mice. The numbers at the top in the D₃-treated lanes indicate fold increase of Sult2A1 mRNAs in the young (3-fold) and old (2-fold) compared to vehicle treatment. The fold increase was calculated from the scanned gel bands as described under Results. 2B) Immunoprecipitation of solubilized liver chromatin fragments to assay for the enrichment of VDR and RXR-α at the IR0 in vitamin D₃-injected 4 month and 20 month old mice. The COX-2 antibody was a negative control.

Figure 3. Sult2A1 gene induction in PCN-injected young and old mice

3A) RT-PCR analysis of the Sult2A1 mRNA induction in the livers of 4-month and 20-month old PCN-injected mice. Numbers at the top indicate the fold induction of Sult2A1 mRNAs after PCN injection compared to the vehicle control. 3B) ChIP analysis of liver chromatin from young and old mice to determine the activity of IR0 to recruit PXR and RXR-α in response to PCN injection.