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Research ArticleCellular and Molecular

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

Aditya D. Joshi, Dwayne E. Carter, Tod A. Harper Jr. and Cornelis J. Elferink
Journal of Pharmacology and Experimental Therapeutics April 2015, 353 (1) 201-212; DOI: https://doi.org/10.1124/jpet.114.222265

Abstract

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated 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 (H2O2)-, thapsigargin-, and ethanol-induced apoptosis in an AhR-dependent manner. Furthermore, suppressing Stc2 expression using RNA interference confirmed that the cytoprotective properties of CA against H2O2, 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 ethanol-induced 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.

Introduction

The liver is an immensely complex organ engaged in unique biochemical functions essential for survival. Liver damage is commonly observed in the clinical setting due to the prevalent use of alcohol, exposure to pharmacological agents, hepatotropic pathogens, and various disease states. Since most forms of liver injury target the hepatocytes, either directly or indirectly, an understanding of the hepatocyte cellular response mechanisms to injury is essential for the development of therapeutic strategies designed to ameliorate liver dysfunction due to injury or disease. The aryl hydrocarbon receptor (AhR) is highly expressed in hepatocytes and is implicated in physiologic liver homeostasis, including cell cycle control and apoptosis (Park et al., 2005; Mitchell et al., 2006; Wu et al., 2007).

The AhR is a cytosolic, ligand-activated transcription factor belonging to the basic helix-loop-helix/Per-Arnt-Sim family of transcription factors. The AhR is predominantly known as a mediator of adaptive and toxic responses to a variety of environmental pollutants, such as 2,3,7,8-tetrachlorodibenzodioxin (TCDD). Ligand activation of the AhR results in nuclear translocation of the receptor where it dimerizes with the AhR nuclear translocator (Arnt), or the recently identified binding partner, the Kruppel-like factor 6 (KLF6) (Legraverend et al., 1982; Hankinson, 1993, 1995; Wilson et al., 2013). The AhR-Arnt dimer regulates the transcription of numerous drug-metabolizing enzymes, including CYP1A1, by binding to xenobiotic response elements (XREs) located in the target gene promoters (Revel et al., 2003; Marlowe and Puga, 2005; Chopra and Schrenk, 2011). The activated AhR-Arnt complex is subsequently regulated by ubiquitin-mediated proteosomal degradation. Studies utilizing AhR-null mice have demonstrated that AhR plays a crucial antiapoptotic role in the liver and lungs (Wu et al., 2007; Abe et al., 2014). However, the downstream AhR signaling cascade responsible for this cytoprotection remains largely unknown despite extensive research on the receptor’s response to exogenous agonists. Recently, endogenous tryptophan catabolites such as 6-formylindolo[3,2-b]carbazole (FICZ) (Wei et al., 1998), l-kynurenine (Opitz et al., 2011), and cinnabarinic acid (CA) (Lowe et al., 2014) have been shown to activate AhR. These proposed endogenous agonists have provided new tools to mechanistically study the physiologic functions of the receptor.

Our laboratory recently identified stanniocalcin 2 (Stc2) as a novel AhR target gene (Harper et al., 2013). Earlier studies in the neuroblastoma N2a cell line linked selective induction of Stc2 with treatments using endoplasmic reticulum (ER) and oxidative stress agents. In isolated primary hepatocytes, AhR-dependent induction of Stc2 coincided with an ER stress response triggered by adenoviral infection (Harper et al., 2013). Although the physiologic mechanism of Stc2 function remains elusive, it is suspected to be a secreted glycophosphoprotein that acts in an autocrine or paracrine manner to impart a protective function in the unfolded protein response and apoptosis (Ito et al., 2004; Wagner and Dimattia, 2006). A recent study suggested that Stc2 conferred cytoprotection against ER stress by suppressing apoptotic cell death through a mechanism involving the PERK-ATF4 signaling cascade (Fazio et al., 2011). Although exposure to the prototypical exogenous AhR agonists TCDD, 3-methylchloranthrene, and β-naphthoflavone failed to induce Stc2 expression in primary hepatocytes (Harper et al., 2013), in the present study, we show that AhR activation by the recently identified endogenous agonist, CA, induces Stc2 expression in isolated mouse primary hepatocytes as well as in vivo. The present study also provides evidence that the suppression of hepatocyte cell death induced by several ER stressors, including H2O2, thapsigargin, and ethanol, is absolutely dependent on CA-inducible AhR-dependent induction of Stc2 expression.

Materials and Methods

Animals and In Vivo Treatments.

Eight- to 10-week-old AhRfl/fl (AhR floxed) and AhRfl/fl/CreAlb [liver-specific AhR conditional knockout (AhR CKO) mice] (Mitchell et al., 2006) female mice were used in compliance with the guidelines of the Institutional Animal Care and Use Committee at the University of Texas Medical Branch. Mice were maintained on a 12-hour light/dark cycle and allowed free access to water and chow. For acute alcohol treatments, ethanol (6 g/kg body weight) was administered through gavage (Zhou et al., 2001). Mice pretreated with CA were given a single intraperitoneal dose of CA at a 12-mg/kg concentration (Mold, 2011; Fazio et al., 2012, 2014). Twelve hours post-CA injection, ethanol (6 g/kg) was administered orally. Mice were sacrificed 12 hours after the ethanol treatment.

Primary Hepatocyte Isolation and Treatments.

Hepatocytes from AhR floxed and AhR CKO were isolated using the collagenase perfusion method as previously described (Park et al., 2005). Isolated primary hepatocytes were plated at a density of 1,000,000 cells/cm2 in Williams’ E medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 5% fetal bovine serum. Cells were treated with 10 or 30 µM CA dissolved in dimethylsulfoxide or 6 nM TCDD for 24 hours. Primary hepatocytes were treated with 300 µM H2O2 for 1 hour or with 3 µM thapsigargin and 100 mM ethanol for 24 hours. For Stc2 overexpression or knockdown studies, primary hepatocytes were transiently transfected with Stc2/pCMV-SPORT6 (Thermo Scientific, Wilmington, DE) or ON-TARGETplus Stc2 small interfering RNA (siRNA; Thermo Scientific), respectively. Metafectene PRO (Biontex, San Diego, CA) was used as a transfection agent. For the cyclohexamide studies, cyclohexamide (10 µg/ml) was added to the medium 1 hour prior to treatment with 30 µM CA. To prevent overt cytotoxicity, cyclohexamide was removed after 6 hours by providing fresh medium supplemented with 30 µM CA under conditions in which CA treatment extended beyond 5 hours.

RNA Isolation and Reverse-Transcription Polymerase Chain Reaction Analysis.

Total RNA from primary hepatocytes or liver tissue was isolated using Trizol (Life Technologies, Carlsbad, CA) according to the manufacturer’s directions. RNA yield was assessed using Nanodrop ND-100 (Thermo Scientific). First-strand cDNA was prepared from 1 µg of RNA using oligo (dT) primer (New England Biolabs, Ipswich, MA) and superscript II reverse polymerase (Life Technologies). Semiquantitative polymerase chain reaction (PCR) was performed using Taq polymerase (Fisher Scientific, Pittsburg, PA) using oligonucleotide primers for mouse Stc2 (forward 5′-TCTGCACAACGCTGGAAAAT-3′, reverse 5′-CTCTGTTGGCTGTTAGGTGATGG-3′) and for mouse CYP1A1 (forward 5′-CGGGACATCACAGACAGCCTCATT-3′, reverse 5′-CCTGCCACTGGTTCACAAAGACAC-3′). Quantitative reverse-transcription PCR (RT-PCR) was performed in the Molecular Genomics Core Facility using Applied Biosystems 7500 Real-Time PCR System (Life Technologies).

Western Blot Analysis.

Whole-cell lysates from primary hepatocytes or liver tissue lysates were prepared in cell lysis buffer (Cell Signaling Technology, Danvers, MA). Proteins were fractionated by SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences, Piscataway, NJ). Rabbit polyclonal antibody was custom generated against C-terminal epitope (283-EWEDEQSEYSDIRR-296) of Stc2 (ProSci Inc., Poway, CA). Fluorescent secondary antibodies were used and were imaged using the Typhoon Trio Variable Mode Imager (GE Healthcare, Waukesha, WI).

Chromatin Immunoprecipitation.

The primary hepatocytes treated (±30 µM CA for 24 hours) were collected and subjected to chromatin immunoprecipitation (ChIP) using the ChIP-IT Express Enzymatic Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. For immunoprecipitation, histone H3 (positive control), IgG (negative control), AhR, and Arnt antibodies were used. Input and immunoprecipitated DNA were PCR amplified using Stc2 (forward 5′-CTCAGTCCATTCGGCCATTGC-3′, reverse 5′-ACTTCTACGGGAGGAAGCGGAG-3′) and CYP1A1 (forward 5′-CTATCTCTTAAACCCCACCCCAA-3′, reverse 5′-CTAAGTATGGTGGAGGAAAGGGTG-3′) primers. PCR product was run on 5% polyacrylamide gel, stained with SYBR green I (Life Technologies) and imaged on Typhoon Trio (GE Healthcare).

Immunofluorescence and Confocal Microscopy to Localize Stc2.

Isolated primary hepatocytes were plated on Laboratory-Tek II chamber slides (Fisher Scientific) at a density of 20,000/1.7 cm2 and treated with dimethylsulfoxide, CA (30 µM), and TCDD (6 nM) for 24 hours. Liver tissues (±12 mg/kg CA for 24 hours) were collected and frozen OCT blocks (Sakura Finetek USA Inc., Torrance, CA) were prepared. Frozen liver tissue sections were prepared using Leica CM1900 cryostat (Leica Biosystems, Buffalo Grove, IL). Primary hepatocytes and tissue sections were fixed with 4% paraformaldehyde for 30 minutes at 4°C. Cells were permeabilized with 0.2% Triton X-100 for 30 minutes and blocked for 1 hour with 5% bovine serum albumin. Cells were treated with either anti-AhR (Enzo Life Sciences, Farmingdale, NY) or anti-Stc2 (ProSci Inc.), and tissue sections were stained with Stc2 antibody (1:1000) overnight at 4°C and treated with Alexa Fluor 488 anti-goat secondary antibody (Life Technologies; 1:100). Anti–pan-cadherin antibody (1:200) was used to label plasma membrane, and mouse anti-KDEL antibody (1:200) was used as an ER marker. Alexa Fluor 594 was used as a secondary antibody to visualize membrane or ER markers. Nuclei were stained with SlowFade Gold with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies). Cells were imaged with a Nikon D-Eclipse C1 inverted confocal microscope (60× oil immersion; Nikon Instruments Inc., Melville, NY) or with a Zeiss Axiovert 200 epifluorescence microscope (Zeiss, Thornwood, NY).

H&E and Oil Red O Staining.

H&E staining was performed by Vel Laboratories (Houston, TX) on paraffin-embedded tissues from mice treated with vehicle, CA, ethanol, and CA + ethanol, whereas Oil Red O staining was done on treated frozen liver tissue sections by Research Histopathology Core at the University of Texas Medical Branch (Galveston, TX). Lipid content was quantified using MetaMorph image analysis software (Molecular Devices, Sunnyvale, CA) as described previously (Clement et al., 2008).

Terminal Deoxynucleotidyl Transferase–Mediated Digoxigenin-Deoxyuridine Nick-End Labeling Assay.

For terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL) assay, isolated primary hepatocytes or mice in vivo were treated with CA as described previously. Alternatively, primary hepatocytes were treated with H2O2, thapsigargin, and ethanol as described earlier. Liver tissues were also collected from mice treated with vehicle, CA, ethanol, and CA + ethanol and processed for cryosectioning. Cells and tissues were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 20 minutes, washed with PBS, and then incubated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 minutes. Fixed and permeabilized slides were incubated with TUNEL reaction mixture provided in an in situ cell death detection kit, tetramethylrhodamine red (Roche, Nutley, NJ), for 1 hour at 37°C. Slides were washed with PBS and then treated with SlowFade Gold with DAPI (Life Technologies) to stain nuclei. Apoptotic nuclei were imaged using a Zeiss Axiovert 200 epifluorescence microscope.

Caspase-3 Assays.

Caspase-3 assays were performed as described previously (Harper et al., 2013). In brief, hepatocytes or tissues were resuspended in lysis buffer [10 mM Tris (pH 7.5), 10 mM NaH2PO4/Na2HPO4 (pH, 7.5), 130 mM NaCl, 1% (v/v) Triton X-100, 10 mM NaPPi]. Cells were homogenized using Dounce homogenizer and centrifuged at 10,000g for 10 minutes. Supernatants were transferred to new microcentrifuge tubes, and 50 µl of supernatant was added to 50 μl of assay buffer/dithiothreitol mix [20 mM PIPES, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% (w/v) CHAPS, 10% (w/v) sucrose (pH 7.2)]. Five microliters of 1 mM fluorogenic caspase-3 substrate, Ac-DEVD-AFC, was added to each sample and allowed to incubate at 37°C for 1 hour. Fluorescence resulting from the cleavage of 7-amino-4-trifluoromethylcoumarin was quantified fluorometrically using a Fluoroskan Ascent fluorometer (Thermo Fisher Scientific, Waltman, MA) with a 400-nm excitation filter and 505-nm emission filter.

Alanine Aminotransferase Assay.

Serum alanine aminotransferase (ALT) assay activity was measured fluorometrically using the Sigma ALT activity assay kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s instructions.

Statistical Analysis.

Data were analyzed by applying the t test using Sigma Plot software (Systat Software, San Jose, CA). Data are represented as the mean ± S.E.M. Differences between the groups are considered statistically significant if the P value is <0.05.

Results

Cinnabarinic Acid Induces Stc2 in an AhR-Dependent Manner.

Our laboratory has identified a novel AhR target gene, Stc2, whose expression is refractory to exogenous AhR agonists (Harper et al., 2013). Recently, an endogenous AhR agonist, CA, was identified (Lowe et al., 2014; Supplemental Fig. 1). We measured the time and dose response of CA on Stc2 expression in isolated primary hepatocytes from AhR-floxed animals. Maximal Stc2 induction was achieved with 30 µM CA treatment for 24 hours (Fig. 1A). The reduced induction at 48 hours suggests that CA is a short-lived AhR agonist. The reduced Stc2 induction detected with 50 µM CA is attributed to a cytotoxic effect observed in the primary hepatocyte cultures at concentrations above 30 µM CA. Consequently, all subsequent experiments used CA concentrations of 30 µM or less. Studies using cyclohexamide (10 µg/ml) to inhibit nascent protein synthesis in primary hepatocytes demonstrated that the Stc2 induction persisted in the absence of new protein synthesis, suggesting that the increased Stc2 expression is a direct AhR-mediated response to CA (Fig. 1A). Interestingly, quantitative RT-PCR of total RNA from AhR-floxed hepatocytes revealed that CYP1A1- and Stc2-induced expression by TCDD and CA was mutually exclusive (Fig. 1B). TCDD only induced CYP1A1 expression, whereas Stc2 induction was only observed in CA-treated cells. The data also confirmed that induced Stc2 expression was refractory to CA treatment in AhR CKO primary hepatocytes, consistent with our previously published finding that inducible Stc2 expression is AhR dependent (Harper et al., 2013). Upon TCDD treatment, AhR CKO primary hepatocyte cultures retained measurable albeit markedly reduced CYP1A1 expression compared with hepatocytes from AhR-floxed mice, which is attributed to the presence of AhR-positive nonparenchymal cells contaminating the cell preparation. Western blotting confirmed that the CA-induced increase in Stc2 mRNA is accompanied by elevated protein expression in AhR-positive hepatocytes, but not in cells isolated from the AhR CKO animals (Fig. 1C).

Fig. 1.
Fig. 1.

CA upregulates Stc2 expression. (A) Isolated primary hepatocytes from AhR-floxed mice were treated with the indicated concentration of CA for 24 hours (left, n = 2) or with 30 µM CA for the indicated time (right) in the absence of cyclohexamide (gray bars, n = 2) or 10 µg/ml cyclohexamide for 6 hours (black bars, n = 1). Cyclohexamide was added to the medium 1 hour prior to CA treatment and was removed after 6 hours by adding fresh medium supplemented with CA. Stc2 mRNA was quantified by RT-PCR and normalized against 18S ribosomal RNA. (B) Isolated primary hepatocytes from AhR-floxed and AhR CKO mice were untreated or treated with vehicle [dimethylsulfoxide (DMSO)] or the indicated concentration of CA or 6 nM TCDD for 24 hours. Quantitative RT-PCR on total RNA was performed to measure Stc2 and CYP1A1 mRNA levels, using 18s ribosomal RNA as a control (n = 3). (C) Western blotting against Stc2 from isolated AhR-floxed and CKO hepatocytes treated with CA or TCDD as described for (B).

Direct Binding of AhR to Stc2 Promoter upon Cinnabarinic Acid Treatment.

The Stc2 promoter contains clusters of putative XREs spanning a 250-bp region located within the first 500 bp upstream of the Stc2 transcription start site. Previous ChIP studies demonstrated that this sequence recruited the AhR in the absence of an exogenous agonist (Harper et al., 2013). Given the ability of CA to directly induce Stc2 expression, we sought to confirm that this was an AhR-dependent process (Fig. 2). ChIP assays targeting the AhR and Arnt protein were performed in isolated primary hepatocytes treated with vehicle or 30 µM CA. Data obtained using PCR primers flanking the XRE cluster in both the Stc2 and CYP1A1 promoters (Harper et al., 2013; Wilson et al., 2013) show that the Stc2 regulatory region, unlike the CYP1A1 promoter, recruits the AhR-Arnt heterodimer as an inducible complex in response to CA (Fig. 2A). We showed previously that TCDD was unable to induce recruitment of the AhR to the Stc2 promoter (Harper et al., 2013), consistent with failure of TCDD to induce Stc2 gene expression. Complementary ChIP studies on hepatocytes isolated from the CKO mice failed to detect a DNA-bound complex, consistent with the conclusion that the inducible complex recruited to the Stc2 regulatory region is the AhR-Arnt heterodimer. ChIP with the H3 and IgG antibodies functioned as positive and negative controls, respectively.

Fig. 2.
Fig. 2.

CA treatment induces AhR binding to the Stc2 promoter. ChIP analysis of AhR and Arnt binding to the Stc2 and CYP1A1 promoters in isolated primary hepatocytes from AhR-floxed (A) and AhR CKO (B) mice treated with vehicle (−) or 30 µM CA (+) for 24 hours. Histone H3 and IgG antibodies were used as positive and negative controls, respectively. PCR primers flanking XRE clusters in the Stc2 and CYP1A1 promoters were used to amplify the precipitated DNA. PCR products were fractionated and visualized on 5% polyacrylamide gels.

Subcellular Localization of Endogenous Stc2.

Several reports speculated that Stc2 is a secreted protein based on findings with the homologous protein Stc1 (McCudden et al., 2002; Wagner and Dimattia, 2006). However, direct evidence for Stc2 secretion has not been documented to date. A recent report using transfected COS cells demonstrated Stc2 localization to the ER and Golgi compartment consistent with a secretory fate (Ito et al., 2004). To determine the subcellular distribution of Stc2 in primary hepatocytes, we performed localization studies using an anti-Stc2 antibody. Immunofluorescence on nonpermeabilized hepatocytes treated with CA revealed punctate staining on the cell surface (Fig. 3). In contrast, CA-treated permeabilized hepatocytes revealed abundant Stc2 staining prominently in the ER, based on colocalization with the ER marker KDEL (Fig. 3). Complementary confocal studies performed on liver sections from AhR-floxed and CKO mice examined the distribution of hepatic Stc2 in vivo. Stc2 expression was undetectable in vehicle-treated AhR-floxed mouse liver, but was readily detectable following CA treatment (Fig. 4). Moreover, Stc2 staining was predominantly present as puncta associated with the plasma membrane based on the overlapping distribution of the membrane marker pan-cadherin (Fig. 4, A–H). In keeping with the staining data obtained in primary hepatocytes, the staining seen in vivo is most likely due to secreted Stc2 protein binding to target sites (e.g., a cell-surface receptor) rather than Stc2 being localized to the intracellular surface of the plasma membrane. The inability to detect Stc2 staining in the ER suggests that Stc2 secretion in vivo may be more efficient, thereby preventing the accumulation of Stc2 observed in primary hepatocyte ER. Furthermore, since Stc2 staining is not observed in the livers of CA-treated CKO mice, consistent with the absence of Stc2 induction, the data reveal that the antibody is Stc2 specific (Fig. 4, I–P).

Fig. 3.
Fig. 3.

Subcellular localization of Stc2. Primary hepatocytes from AhR-floxed mice were treated with vehicle [dimethylsulfoxide (DMSO)] or 30 µM CA for 24 hours and fixed with 4% paraformaldehyde in the absence (A–H) and presence (I–P) of 0.2% Triton X-100. Cells were probed with an anti-Stc2 primary antibody and an Alexa Fluor 488 secondary antibody (green), or antibodies against pan-cadherin (C and G) or KDEL (K and O) and the Alexa Fluor 594 secondary antibody (red). Images were acquired using a Nikon D-Eclipse C1 inverted confocal microscope (60× oil immersion). DAPI was used to stain nuclei (blue).

Fig. 4.
Fig. 4.

Stc2 expression and subcellular localization in mouse liver. AhR-floxed (A–H) and CKO (I–P) mice were treated with vehicle (saline) or CA (12 mg/kg) for 24 hours. Immunofluorescence was performed on frozen liver sections probing for Stc2 (green) and pan-cadherin (red) as a membrane marker. DAPI was used to stain nuclei. A Nikon D-Eclipse C1 inverted confocal microscope was used to capture images.

Cinnabarinic Acid–Dependent AhR Mediated Cell Protection against Oxidative/ER Stress–Induced Apoptosis.

Previous studies established that the AhR promotes cell survival in response to intrinsic apoptotic stimuli, including H2O2, UV radiation, or viral infection (Wu et al., 2007; Harper et al., 2013). We sought to demonstrate that CA could protect hepatocytes against intrinsic apoptotic cell death induced by H2O2 (oxidative stressor), thapsigargin (ER stressor), or ethanol (oxidative and ER stressor) in an AhR-dependent mechanism. Primary hepatocytes isolated from AhR-floxed and CKO mice were pretreated with vehicle, TCDD, or CA and subjected to the aforementioned apoptotic stimuli (Fig. 5). Apoptosis was assessed by measuring caspase-3 activity. CA conferred cytoprotection upon AhR-positive hepatocytes (Fig. 5, A–C), but not AhR-negative cells (Fig. 5, D–F). In contrast, TCDD completely failed to provide protection irrespective of the receptor status. It should be noted that CA was not able to protect against extrinsic apoptotic signaling induced by tumor necrosis factor α and Fas ligand (Supplemental Fig. 2), consistent with the earlier finding that the AhR promotes apoptosis in response to extrinsic cell death signals (Park et al., 2005; Wu et al., 2007).

Fig. 5.
Fig. 5.

CA-induced AhR activation protects against ER/oxidative stress–mediated apoptosis. Isolated primary hepatocytes from AhR-floxed (A–C) and AhR CKO (D–F) mice were treated with vehicle [dimethylsulfoxide (DMSO)], 10 µM CA, 30 µM CA, or 6 nM TCDD for 24 hours before exposure to 300 µM H2O2 (A and D), 3 µM thapsigargin (B and E), or 100 mM ethanol (C and F). Fluorometric caspase-3 assays were performed on total cell lysates using the caspase-3 substrate, Ac-DEVD-AFC. Caspase-3 activity is normalized to total protein concentrations and to DMSO only–treated primary hepatocytes. Data are represented as the mean ± S.E.M. from three independent experiments (*P < 0.05).

Complementary studies examining DNA fragmentation using the TUNEL assay were performed to verify the antiapoptotic characteristic of the CA-induced AhR (Fig. 6). Primary hepatocytes isolated from AhR-floxed and CKO mice were treated with H2O2, thapsigargin, or ethanol in the presence and absence of CA as described earlier. AhR expressing hepatocytes pretreated with CA showed significantly fewer TUNEL-positive nuclei following exposure to H2O2 (Fig. 6, C and D), thapsigargin (Fig. 6, E and F), and ethanol (Fig. 6, G and H). Evidence of apoptosis is negligible in vehicle-treated cells (Fig. 6A) or cells exposed to CA alone (Fig. 6B). Quantitation of the TUNEL-positive nuclei revealed that the 35–70% apoptosis observed in AhR-positive hepatocytes treated with H2O2, thapsigargin, or ethanol was markedly reduced (≈5%) in cultures pretreated with CA (Fig. 6, Q–S). In contrast, CA was completely ineffective in protecting CKO hepatocytes from the intrinsic death signals used (Fig. 6, I–P and T–V). It is also noteworthy that hepatocyte viability was substantially diminished in the CKO cell cultures when compared with AhR-positive cells after treatment with H2O2, thapsigargin, or ethanol. This suggests that, even in the absence of exogenous CA, the receptor status confers some inherent cytoprotection, presumably due to endogenous AhR activation.

Fig. 6.
Fig. 6.

TUNEL staining reveals AhR-dependent cytoprotection following CA treatment. Primary hepatocytes from AhR-floxed (A–H) and CKO (I–P) mice were pretreated with vehicle [dimethylsulfoxide (DMSO)] or 30 µM CA for 24 hours. Hepatocytes were subjected to 300 µM H2O2 for 1 hour or 3 µM thapsigargin (thap) or 100 mM ethanol for 24 hours prior to fixation in 4% paraformaldehyde. Cells were treated with tetramethylrhodamine red TUNEL reaction mixture (Roche) to label DNA strand breaks and detect apoptotic nuclei (red). DAPI was used to stain nuclei. Images were acquired using a Zeiss Axiovert 200 epifluorescence microscope. Quantitation of apoptotic cells was from three to four independent experiments by counting TUNEL-positive nuclei as a percentage of total nuclei in four to five random fields of AhR-floxed (Q–S) and CKO (T–V) hepatocytes. *Statistically significant difference compared with DMSO-treated (P < 0.05). Data represented as the mean ± S.E.M.

Stc2 Directly Contributes to Cytoprotection against ER/Oxidative Stress.

Stc2 expression was implicated in thapsigargin- and H2O2-induced stress in N2a and PC12 cell lines (Ito et al., 2004). The results are consistent with the hypothesis that AhR-mediated induction of Stc2 by CA is responsible for cytoprotection. To test this directly, we used exogenous CA, recombinant Stc2 expression, and RNA interference to modulate Stc2 expression in hepatocytes and evaluate their susceptibility to intrinsic apoptotic signaling. Western blotting measured the level of Stc2 protein expression under the various experimental conditions (Fig. 7A). The data obtained using caspase-3 activity to measure apoptosis show that ectopic overexpression of a recombinant Stc2 protein protected primary hepatocytes from H2O2-induced apoptosis, whereas transfection with the empty vector (pCMV-SPORT6) failed to provide cytoprotection (Fig. 7B). In the absence of CA, hepatocytes transfected with siRNA designed to suppress Stc2 expression were substantially more susceptible to H2O2-, thapsigargin-, or ethanol-induced apoptosis than cells transfected with a scrambled (control) siRNA oligonucleotide (Fig. 7, C–E). Significantly, Stc2 knockdown also rendered AhR-positive hepatocytes susceptible to apoptosis even in the presence of CA, indicating that the cytoprotective phenotype was absolutely contingent on Stc2 expression. As expected, under conditions that preserved Stc2 expression (CA alone or with the scrambled siRNA), CA enhanced cell survival following exposure to the cell death cues.

Fig. 7.
Fig. 7.

Stc2 expression is required for cell survival. (A) Primary hepatocytes isolated from AhR-floxed and AhR CKO mice were treated with vehicle [dimethylsulfoxide (DMSO)], 30 µM CA, transiently transfected with a construct expressing recombinant Stc2 (Stc2/pCMV-SPORT6), or transfected with a siRNA targeting Stc2. After 24 hours, Western blotting on total cell lysates was used to monitor Stc2 protein expression. Arrows identify the Stc2 protein band migrating above a nonspecific band. Actin was used as a loading control. (B) Primary hepatocytes from AhR-floxed mice were transfected with a control vector (pCMV-SPORT6) or a vector encoding a recombinant Stc2 (Stc2/pCMV-SPORT6) for 24 hours, followed by treatment with 300 µM H2O2 for 1 hour and assayed for caspase-3. Graph represents the mean ± S.E.M. of caspase-3 assays from three independent experiments (*P < 0.05). (C–E) Isolated AhR-floxed primary hepatocytes were transfected with a control siRNA (scrambled) or Stc2 siRNA in the absence and presence of 30 µM CA for 24 hours. Hepatocytes were subsequently treated with 300 µM H2O2 (C), 3 µM thapsigargin (D), or 100 mM ethanol (E). Fluorometric caspase-3 assays were performed and normalized to total protein concentration in three to four independent experiments, where statistically significant variance from the indicated treatment alone (DMSO + H2O2, thapsigargin, or ethanol) is identified by an asterisk (mean ± S.E.M., *P < 0.05).

Cinnabarinic Acid Protects against Ethanol-Induced Apoptosis In Vivo.

We next evaluated the capacity of CA to protect against apoptosis in vivo using a mouse model of acute alcohol hepatotoxicity (Zhou et al., 2001). RT-PCR analysis of liver lysate revealed that a 12-mg/kg dose of CA administered by intraperitoneal injection induced a maximal 4-fold induction of Stc2 after 24 hours (Supplemental Fig. 3). Although Fazio et al. (2012) determined that basal CA levels in several rat tissues ranged from 0 to 60 pg/mg, the CA levels could be dramatically induced (≈160 pg/mg) in tissues subjected to inflammatory conditions, indicative of a broad physiologic concentration range. Likewise, Lowe et al. (2014) showed that inflammatory agents induced CA concentrations in human peripheral blood mononuclear cell cultures to ≈300 ng/ml. The dearth of pharmacokinetic information on exogenously administered CA precludes assessment of the experimental CA concentration achieved in vivo over the 24-hour study period used here. The CA concentration, however, is not envisioned to be grossly supraphysiological. As expected, Stc2 mRNA levels remained essentially unchanged in AhR CKO mouse livers following CA treatment. Western blotting confirmed that the CA-induced increase in liver Stc2 mRNA detected in vivo was supported by an increase in protein expression, but only in the AhR-floxed mouse livers. Administration of TCDD (20 µg/kg by gavage) did not increase Stc2 expression, consistent with the results obtained using primary hepatocytes. No overtly adverse phenotypic changes were observed after in vivo CA treatment. To further investigate the cytoprotective role of CA in vivo, we used an acute alcohol hepatotoxicity mouse model (Zhou et al., 2001). AhR-floxed and CKO mice were treated with a single dose of 6 mg/kg ethanol orally for 12 hours to induce apoptosis, in the absence or presence of CA pretreatment. Control mice were treated with vehicle (saline) or 12 mg/kg CA. Both caspase-3 and TUNEL assays demonstrated that CA pretreatment protected AhR-floxed animals from ethanol-induced hepatic cell death (Fig. 8, A and C–F, respectively). The cytoprotective effect of CA was not observed in the AhR CKO mouse livers (Fig. 8, B and G–J). The modest increase in caspase-3 activity detected in the CA-treated mouse liver tissues (Fig. 8, A and B) was not statistically different from the saline-treated controls, in keeping with the absence of TUNEL positivity in the liver sections (Fig. 8, D and H). Histologic evaluation of the livers revealed microvesicular steatosis in the ethanol-treated mice (Fig. 9, A–H). The presence of steatosis was confirmed using Oil Red O staining which specifically detects lipid droplets. Significantly, CA pretreatment prevented onset of the steatotic microvesicles induced by acute ethanol treatment, reinforcing the prior findings for CA’s protective role. Accordingly, this cytoprotection was not observed in the CKO mouse livers, which instead exhibited extensive microvesicular streatosis irrespective of CA pretreatment (Fig. 9, I–P; Supplemental Fig. 4). Serum ALT assays confirmed that the ethanol treatment resulted in acute liver damage that was significantly attenuated by CA pretreatment in the AhR-positive livers (Fig. 9Q), but not the CKO livers (Fig. 9R). Collectively, the data illustrate that the AhR in response to CA can confer cytoprotection in vivo.

Fig. 8.
Fig. 8.

CA protects against ethanol-induced liver apoptosis in vivo. AhR-floxed (A) and AhR CKO (B) mice were treated intraperitoneally with saline, 12 mg/kg CA, 6 g/kg ethanol administered by gavage, or with CA for 12 hours prior to ethanol for 12 hours. Liver homogenates were subjected to caspase-3 fluorometric assays. Results shown are the mean ± S.E.M. from three independent experiments (each treatment group contained three mice, * P < 0.05 difference between ethanol-treated and CA + ethanol–treated samples). Data are normalized to caspase activity in the saline-treated groups. TUNEL assays were performed on liver sections from AhR-floxed (C–F) and CKO (G–J) mice treated with saline, CA, ethanol, and CA + ethanol, with all hepatic nuclei (DAPI) and apoptotic nuclei (red) visualized, respectively.

Fig. 9.
Fig. 9.

CA protects against acute alcohol hepatotoxicity. H&E staining of liver sections from AhR-floxed (A–D) and AhR CKO (I–L) mice treated with vehicle (saline), 12 mg/kg CA, 6 g/kg ethanol, or CA pretreatment followed by ethanol as described in the Fig. 8 legend. Oil Red O staining was used to monitor steatosis in AhR-floxed (E–H) and AhR CKO (M–P) mouse livers following acute ethanol treatment. Representative images are shown for each of the treatments (n = 3). ALT enzyme levels were measured using a Sigma-Aldrich diagnostic kit in serum collected from AhR-floxed (Q) and CKO mice (R). Three mice were used for each treatment group (mean ± S.E.M., *P < 0.05).

Discussion

Liver disease is a common clinical malady due to excessive alcohol use, pharmacological and toxicological agents, viral infections, as well as various other pathogenic factors. Hepatocytes, comprising the liver parenchyma, constitute about 75–80% of liver cytoplasmic mass and is the major target of liver injury. Apoptosis of hepatocytes is rare in healthy livers (1–5 apoptotic cells/10,000 hepatocytes) (Schulte-Hermann et al., 1995), but in certain liver diseases, such as alcohol liver disease, cirrhosis, viral hepatitis, fulminant hepatitis, hepatocellular carcinoma, nonalcoholic steatohepatitis, and cholestatic liver disease, pronounced induction of hepatocyte apoptosis is associated with disease (Benedetti et al., 1988; Goldin et al., 1993; Krammer, 1996; Feldmann, 1997; Kondo et al., 1997; Sodeman et al., 2000; Guicciardi and Gores, 2002; Feldstein et al., 2003; Ribeiro et al., 2004; Park et al., 2005; Madesh et al., 2009). Previous AhR studies have focused primarily on the responses to exogenous receptor agonists—including the polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons—with correspondingly few studies examining the receptor’s physiologic role in the context of endogenous signaling. However, these studies have shown that the AhR can influence cell survival during tissue homeostasis. Specifically, the AhR protects against apoptosis induced by intrinsic cell death signaling (Gonzalez and Fernandez-Salguero, 1998; Wu et al., 2007; Marlowe et al., 2008) while predisposing cells to an apoptotic fate in response to extrinsic cues (Park et al., 2005). The precise molecular basis for the latter remains largely unexplored. This report provides compelling evidence for CA-induced AhR-mediated expression of Stc2 as a critical modulator in promoting hepatocyte survival in the face of multiple intrinsic cell death stimuli, both in cultured primary cells and, more importantly, in vivo. The ability to protect the liver from alcohol-induced hepatotoxicity is a novel finding with potential clinical significance.

To date, several tryptophan derivatives have been implicated as novel endogenous AhR agonists, including 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), tryptamine, indirubin, FICZ, and kynurenine (DiNatale et al., 2010; Denison et al., 2011). FICZ is a high-affinity AhR agonist (Kd = 7 × 10−11 M) that activates the receptor nearly as well as the prototypical exogenous agonist TCDD (Wei et al., 1998). Kynurenine is another endogenous AhR agonist that binds to the AhR with a Kd of 4 µM and is capable of inducing the CYP1A1 AhR target gene (Opitz et al., 2011). We did not observe induction of Stc2 by FICZ in the primary hepatocytes and noticed only modest and inconsistent upregulation of Stc2 mRNA with kynurenine (data not shown). The inconsistency might be due to spontaneous oxidation of kynurenine to 3-hydroxykynurenine and subsequently 3-hydroxyanthranilic acid, neither of which is an AhR ligand (Lowe et al., 2014). CA is an endogenous tryptophan metabolite and a byproduct of the kynurenine pathway, derived through the condensation of two molecules of 3-hydroxyanthranilic acid. There are reports of enzymatic processes involving cinnabarinate synthase in the liver (Rao and Vaidyanathan, 1966), ceruloplasmin in the bacteria Pycnoporus cinnabarinus, and fungal virulence factor laccase (Eggert, 1997) that lead to the formation of CA. Evidence also exists for enhanced CA production by superoxide anion scavengers such as superoxide dismutase (Dykens et al., 1987) and the nonenzymatic oxidation of 3-hydroxyanthranilic acid to CA (Hiramatsu et al., 2008). Although the precise mechanism responsible for CA formation in vivo remains obscure, CA was recently identified as a bona fide AhR agonist (Lowe et al., 2014). Functionally, little is known about CA apart from its involvement in activating the differentiation of T cells and expression of interleukin-22 (Lowe et al., 2014).

The ChIP data revealed that CA treatment induced AhR–Arnt heterodimer binding directly to a 257-bp region of the Stc2 promoter located between −450 and −193—a sequence harboring a cluster of 8 putative XREs. We recently demonstrated that TCDD was unable to induce recruitment of the AhR to the Stc2 promoter (Harper et al., 2013). Surprisingly, CA did not recruit the AhR-Arnt complex to the CYP1A1 enhancer (encompassing a cluster of XREs), nor did it induce CYP1A1 mRNA expression (Figs. 1 and 2). Hence, the results in primary hepatocytes revealed a striking reciprocity between the CYP1A1 and Stc2 gene transcriptional response following CA and TCDD treatment. It is tempting to speculate that this reflects recruitment of different cofactors/coactivators similar to that of the type 2 nuclear hormone receptor as well as the ErB/HER receptor (Wurtz et al., 1996; Resche-Rigon and Gronemeyer, 1998; Saeki et al., 2009). Conceivably, the agonist-specific transcriptional responses targeting Stc2 and CYP1A1 expression are due to distinct epigenetic chromatin modifications. It was reported that TCDD treatment resulted in acetylation of histone H3 at lysines 9 and 14, acetylation of histone H4, and trimethylation of histone H3 at lysine 4 at the CYP1A1 promoter (Beedanagari et al., 2010). The epigenetic changes brought about by CA may differ, resulting in preferential Stc2 expression in hepatocytes. Future studies focusing on this issue may prove most insightful.

Previous studies suggested that Stc2 plays a cytoprotective role during thapsigargin- and tunicamycin-induced ER stress in N2a and PC12 cell lines (Ito et al., 2004). Fazio et al. (2011) reported that Stc2 alters PERK signaling and reduces apoptosis, autophagy, and cellular injury in cerulean-induced pancreatitis. Our results demonstrated that AhR activation by CA induces expression of Stc2 and confers cytoprotection against ER/oxidative stress–induced apoptosis in primary hepatocytes and the liver in vivo. We specifically chose three apoptotic agents that trigger ER and/or oxidative stress by different mechanisms. Thapsigargin, a noncompetitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase, induces ER stress; hydrogen peroxide (H2O2) generates reactive oxygen species and subsequent oxidative damage; and ethanol is metabolized by CYP2E1 to generate reactive oxygen species (Jarvelainen et al., 1997; Su et al., 1998; Mi et al., 2000; Zhou et al., 2001; Purohit et al., 2009), resulting in glutathione depletion and oxidative stress (Meister, 1974, 1988a,b; Tazi et al., 2007). Oxidative stress leads to phosphoinositide-specific phospholipase C activation and elevated inositol triphosphate levels and Ca2+ mobilization involving the inositol triphosphate receptor. The Ca2+ release induces caspase-mediated apoptosis (Berridge et al., 1998; Wu and Cederbaum, 2003; Ribeiro et al., 2004; Madesh et al., 2005, 2009; Albano, 2006). Potentially fatty acid ethyl esters, nonoxidative metabolites of ethanol, may also induce apoptosis via an intrinsic cell death pathway (Wu et al., 2006). Therefore, the evidence suggests that AhR-induced Stc2 expression provides a general defense mechanism against a broad range of insults. However, the process appears to be restricted to the intrinsic apoptotic pathway, because Stc2 expression failed to confer protection against extrinsic death signals, including Fas and tumor necrosis factor α (Supplemental Fig. 2), in keeping with previous observations (Park et al., 2005; Wu et al., 2007). Although the work by Wu et al. (2007) associated the receptor’s protective function with phosphatidylinositol 3–OH kinase–Akt/protein kinase B activation, a recent study suggested that Stc2 provided cell protection by negatively regulating intracellular store-operated calcium entry, thus limiting the free Ca2+ concentration necessary for initiation of apoptosis (Zeiger et al., 2011). Consequently, the AhR may influence multiple signaling pathways conducive to cell survival.

Our immunohistological data clearly indicate that, in addition to localization to the ER (Fig. 3, I–P), Stc2 is also detected on the extracellular surface of the plasma membrane (Figs. 3, A–H, and 4, A–H). These findings are the first to establish that Stc2 is indeed a secreted protein, and the punctate staining pattern on the membrane in both primary hepatocytes and in liver sections is suggestive of Stc2 binding to discrete receptor sites. Since Stc2 surface staining was exclusively observed on hepatocytes transiently transfected with the Stc2 expression construct, we surmised that Stc2 functions in an autocrine rather than a paracrine capacity (data not shown). However, since the signaling mechanism initiated by secreted Stc2 remains obscure, future studies centered on identifying the putative Stc2 surface receptor should prove informative.

In summary, this study mechanistically describes a physiologic role for the AhR in apoptosis by demonstrating the functional importance of Stc2, a recently identified AhR target gene distinct from the traditional targets involved in xenobiotic regulation. The evidence confirms that Stc2 induction is both AhR dependent and agonist specific. CA, rather than TCDD, stimulates Stc2 expression. Moreover, the data establish that the receptor-mediated increase in Stc2 expression is essential for cytoprotection of primary hepatocytes and the liver in vivo following exposure to apoptotic stimuli that trigger intrinsic cell death pathways. It is conceivable that these observations can be leveraged in the future, into clinically relevant therapeutic strategies designed to protect the liver against injury from various insults.

Authorship Contributions

Participated in research design: Joshi, Carter, Elferink.

Conducted experiments: Joshi, Carter, Harper.

Performed data analysis: Joshi, Carter, Elferink.

Wrote or contributed to the writing of the manuscript: Joshi, Carter, Elferink.

Footnotes

    • Received December 18, 2014.
    • Accepted February 10, 2015.

  • A.D.J. and D.E.C. contributed equally to this work.

  • This work was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grants R01-ES007800 and P30-ES006676 (to C.J.E.)].

  • dx.doi.org/10.1124/jpet.114.222265.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

AhR
aryl hydrocarbon receptor
ALT
alanine aminotransferase
Arnt
AhR nuclear translocator
CA
cinnabarinic acid
ChIP
chromatin immunoprecipitation
CKO
conditional knockout
DAPI
4′,6-diamidino-2-phenylindole
ER
endoplasmic reticulum
FICZ
6-formylindolo[3,2-b]carbazole
ITE
2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
RT-PCR
reverse-transcription polymerase chain reaction
siRNA
small interfering RNA
Stc2
stanniocalcin 2
TCDD/dioxin
2,3,7,8-tetrachlorodibenzodioxin
TUNEL
terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling
XRE
xenobiotic response element

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Research ArticleCellular and Molecular

AhR-Driven Stc2 Expression Confers Cytoprotection

Aditya D. Joshi, Dwayne E. Carter, Tod A. Harper and Cornelis J. Elferink
Journal of Pharmacology and Experimental Therapeutics April 1, 2015, 353 (1) 201-212; DOI: https://doi.org/10.1124/jpet.114.222265
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