Aryl Hydrocarbon Receptor–Dependent Stanniocalcin 2 Induction by Cinnabarinic Acid Provides Cytoprotection against Endoplasmic Reticulum and Oxidative Stress

Abstract

The aryl hydrocarbon receptor (AhR) is a cytosolic ligandactivated transcription factor historically known for its role in xenobiotic metabolism. Although AhR activity has previously been shown to play a cytoprotective role against intrinsic apoptotic stimuli, the underlying mechanism by which AhR confers cytoprotection against apoptosis is largely unknown. Here, we demonstrate that activation of AhR by the tryptophan catabolite cinnabarinic acid (CA) directly upregulates expression of stanniocalcin 2 (Stc2) to elicit cytoprotection against apoptosis induced by endoplasmic reticulum stress and oxidative stress. Chromatin immunoprecipitation studies demonstrated that CA treatment induces direct AhR binding to a region of the Stc2 promoter containing multiple xenobiotic response elements. Using isolated primary hepatocytes from AhR wild-type (AhR floxed) and liver-specific AhR conditional knockout mice, we showed that pretreatment with CA conferred cytoprotection against hydrogen peroxide (H 2 O 2 )-, thapsigargin-, and ethanolinduced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H 2 O 2 , thapsigargin, and ethanol injury were absolutely dependent on Stc2. Immunochemistry revealed the presence of Stc2 in the endoplasmic reticulum and on the cell surface, consistent with Stc2 secretion and autocrine and/or paracrine signaling. Finally, in vivo data using a mouse model of acute alcohol hepatotoxicity demonstrated that CA provided cytoprotection against ethanolinduced apoptosis, hepatic microvesicular steatosis, and liver injury. Collectively, our data uncovered a novel mechanism for AhR-mediated cytoprotection in the liver that is dependent on CA-induced Stc2 activity.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation

“…Although previous reports have suggested that AhR mediates ROS generation in other tissues such as hepatocytes 48) , we failed to detect the involvement of AhR in indoxyl sulfate-triggered oxidative stress in the vascular endothelium. We obtained similar findings using 3MC, another AhR ligand, in the endothelial model (data not shown).…”

And now for something completely different: Diversity in ligand-dependent activation of Ah receptor responses

“…To date, although ligand (TCDD)-dependent alterations in AhR structure have been observed using in vitro synthesized [ 35 S]-labeled AhR and proteolysis approaches [64], ligand-specific differences in overall AhR and/or ARNT structure have not yet been reported. However, a recent study was one of the first to clearly demonstrate AhR ligand-specific differential gene induction in a single cell type [65]. Stannocalcin 2 (Stc2) is a gene whose promoter contains numerous DREs [56,65], however, while classical AhR agonists (TCDD, 3MC and BNF) stimulate AhR binding to DREs upstream of CYP1A1 (measured using ChIP analysis) and induced CYP1A1 gene expression in primary hepatocytes, they failed to stimulate Stc2 gene expression or ligand-dependent binding of AhR to Stc2 promoter DREs [56,65].…”

“…However, a recent study was one of the first to clearly demonstrate AhR ligand-specific differential gene induction in a single cell type [65]. Stannocalcin 2 (Stc2) is a gene whose promoter contains numerous DREs [56,65], however, while classical AhR agonists (TCDD, 3MC and BNF) stimulate AhR binding to DREs upstream of CYP1A1 (measured using ChIP analysis) and induced CYP1A1 gene expression in primary hepatocytes, they failed to stimulate Stc2 gene expression or ligand-dependent binding of AhR to Stc2 promoter DREs [56,65]. In contrast, cinnabarinic acid, a newly identified tryptophan-derived AhR agonist [66], stimulated both AhR binding to the Stc2 promoter DREs and Stc2 gene expression, but failed to stimulate AhR binding to CYP1A1 DREs or induce CYP1A1 gene expression [65].…”

“…Stannocalcin 2 (Stc2) is a gene whose promoter contains numerous DREs [56,65], however, while classical AhR agonists (TCDD, 3MC and BNF) stimulate AhR binding to DREs upstream of CYP1A1 (measured using ChIP analysis) and induced CYP1A1 gene expression in primary hepatocytes, they failed to stimulate Stc2 gene expression or ligand-dependent binding of AhR to Stc2 promoter DREs [56,65]. In contrast, cinnabarinic acid, a newly identified tryptophan-derived AhR agonist [66], stimulated both AhR binding to the Stc2 promoter DREs and Stc2 gene expression, but failed to stimulate AhR binding to CYP1A1 DREs or induce CYP1A1 gene expression [65]. While details of the mechanism(s) responsible for the differential ligand responses of CYP1A1 and Stc2 genes remain to be elucidated, these results are consistent with ligand-selective differences in AhR gene expression and are suggestive of ligand-selective differences in AhR structure/function.…”

Indole and Tryptophan Metabolism: Endogenous and Dietary Routes to Ah Receptor Activation

“…4). Recently, CA also has been shown to play a cytoprotective role in endoplasmic reticulum or oxidative stress-induced apoptosis through AHR-dependent induction of stanniocalcin 2 (Stc2) (Joshi et al, 2015). Further investigation of the kynurenine pathway may lead to the identification of additional endogenous AHR ligands.…”