12 SEROTONIN MODULATES ARYL HYDROCARBON RECEPTOR (AHR) ACTIVATION IN THE INTESTINE BY INTERFERING WITH CYP1A1-MEDIATED CLEARANCE OF AHR LIGANDS

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter and hormone that has many important physiological functions throughout the body. In the gastro-intestinal tract, 5-HT regulates electrolyte and fluid homeostasis, visceral sensation, motility and modulates immune responses. After its release by enterochromaffin cells, 5-HT is transported intracellularly via the Na+/Cl- dependent serotonin transporter (SLC6A4, SERT). We have previously shown that 5-HT activates the aryl hydrocarbon receptor (AhR) in intestinal epithelial cells (IECs) via a SERT-dependent mechanism. AhR is a nuclear receptor that binds both host and bacterial tryptophan metabolites to regulate physiological processes in the intestine including xenobiotic detoxification and immune modulation. The canonical gene target of AhR is CYP1A1, a cytochrome P450 enzyme, involved in phase I xenobiotic and drug metabolism in the intestine. AhR activation by exogenous agonists has been shown to be protective against experimental colitis. Whether 5-HT activates AhR as a ligand or via an alternate mechanism is not known. Models: The human IEC line Caco-2 was used as an in vitro model; wild-type (WT) and SERT knockout (KO) mice were used for in vivo studies. Our data demonstrated that 5-HT potentiated the effects of AhR ligands, supporting an indirect mechanism of AhR activation. This was evident by: 1) Synergistic induction of CYP1A1 expression in Caco-2 cells when co-treated with 5-HT (10 μM) and the AhR ligand 6-formylindolo [3,2-β]carbazole (FICZ 10 nM); 2) Absence of CYP1A1 induction by 5-HT in culture medium devoid of AhR ligands; 3) Marked induction of CYP1A1 expression in response to oral administration of AhR ligand, β-napthoflavone (β-NF; 50 mg/kg) or feeding an indole-3-carbinol (I3C) supplemented diet (200 ppm, 3 wks) in WT but not signifcantly in SERT KO mice. Interestingly, interference with CYP1A1-mediated clearance of AhR ligands has been shown to be an alternative mechanism for AhR activation, as opposed to direct binding to AhR as a ligand. To examine whether 5-HT affects CYP1A1 activity, CYP1A1-mediated metabolism of the pro-luciferin substrate Luc-CEE in the presence or absence of 5-HT was measured. 5-HT inhibited both recombinant CYP1A1 activity and basal CYP1A1 activity in Caco-2 cells (~70%, p<0.05). LC-MS/MS analysis showed that ~90% of 5-HT was metabolized after 1 h of incubation with recombinant CYP1A1, indicating that 5-HT may be a substrate of CYP1A1. Our studies provide evidence for a novel mechanism by which 5-HT can influence the ability of AhR ligands to activate the receptor in IECs. This crosstalk between the serotonergic and AhR pathways may have implications in pathological conditions e.g. inflammatory bowel diseases in which both serotonergic and AhR signaling is dysregulated. (Supported by CCFA, NIH & Vet Affairs).