The Molecular Mechanism of Urolithin in Anti-Aging and Vitamin D Pathways

Mediated by the nuclear vitamin D receptor (VDR), the hormonally active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25D), is known to regulate expression of genes impacting calcium and phosphorus metabolism, the immune system, and behavior. Urolithin A (UA), a nutrient metabolite derived from pomegranate, possibly acting through AMP kinase (AMPK) signaling, supports longevity in C. elegans by inducing oxidative damage-reversing genes. We show herein that UA enhances transcriptional actions of 1,25D driven by co-transfected vitamin D responsive elements (VDREs), and dissection of this genomic effect in cell culture reveals: 1) UA concentration-dependency, 2) occurrence with isolated natural VDREs, 3) nuclear receptor selectivity for VDR over ER, LXR and RXR, and 4) significant 8- to 13-fold UA-augmentation of 1,25D-dependent mRNA encoding the widely expressed 1,25D-detoxification enzyme, CYP24A1, a benchmark vitamin D target gene. Relevant to potential behavioral effects of vitamin D, UA elicits enhancement of 1,25D-dependent mRNA encoding tryptophan hydroxylase-2 (TPH2), the serotonergic neuron-expressed initial enzyme in tryptophan metabolism to serotonin. Employing quantitative real time-PCR, we demonstrate that TPH2 mRNA is induced 1.9-fold by 10 nM 1,25D treatment in culture of differentiated rat serotonergic raphe (RN46A-B14) cells, an effect magnified 2.5-fold via supplementation with 20 mM UA. This potentiation of 1,25D-induced TPH2 mRNA by UA is followed by a 3.1- to 3.7-fold increase in serotonin concentration in culture medium from the pertinent neuronal cell line, RN46A-B14. These results are consistent with the concept that two natural nutrient metabolites, UA from pomegranate and 1,25D from sunlight/vitamin D, likely acting via AMPK and VDR, respectively, cooperate mechanistically to effect VDRE-mediated regulation of gene expression in neuroendocrine cells. Recent evidence also suggests that vitamin D may possess antioxidant activity, especially in the presence of certain nutraceuticals such as curcumin or UA. The transcription factor, Nrf-2, controls the expression of antioxidant response target genes. We also investigated the potential relationship between vitamin D signaling and the Nrf-2 protein. The activity of the vitamin D receptor was evaluated in the presence of variant Nrf-2 levels and multiple concentrations of 1,25D and UA to assess possible crosstalk in regulation of anti-oxidation pathways. Taken together, the results presented herein suggest that over the course of evolution, derivatives of two environmental factors, namely sunlight and pomegranate, appear to have been co-opted for their ability to support the serotonergic nervous system and perhaps to regulate anti-oxidation pathways.