Transcriptional Regulation of Math1 by Aryl Hydrocarbon Receptor: Effect on Math1⁺ Progenitor Cells in Mouse Small Intestine

Abstract

The physiological roles of aryl hydrocarbon receptor (AhR) in the small intestine have been revealed as immunomodulatory and barrier functions. However, its contributions to cell fate regulation are incompletely understood. The Notch-activated signaling cascade is a central component of intestinal cell fate determinations. The lateral inhibitory mechanism governed by Notch directs cell fates toward distinct cell lineages (i.e., absorptive and secretory cell lineages) through its downstream effector, mouse atonal homolog 1 (MATH1). An investigation employing cell lines and intestinal crypt cells revealed that AhR regulates Math1 expression in a xenobiotic response element (XRE)-dependent manner. The AhR-Math1 axis was further addressed using intestinal organoids, where AhR-Math1 and HES1-Math1 axes appeared to coexist within the underlying Math1 transcriptional machinery. When the HES1-Math1 axis was pharmacologically suppressed, β-naphthoflavone-mediated AhR activation increased the number of goblet and Math1⁺ progenitor cells in the organoids. The same pharmacological dissection of the AhR-Math1 axis was applied in vivo, demonstrating an enhanced number of Math1⁺ progenitor cells in the small intestine following AhR activation. We report here that AhR-Math1 is a direct transcriptional axis with effects on Math1⁺ progenitor cells in the small intestine, highlighting a novel molecular basis for fine-tuning Notch-mediated cell fate regulation.

INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that belongs to the basic helix-loop-helix/PER-aryl hydrocarbon receptor nuclear translocator (ARNT)-SIM family. In the basal state, AhR forms a complex with several partners in the cytoplasm of cells. When activated by a ligand, AhR releases the specific partners, then translocates to the nucleus and dimerizes with ARNT, another basic helix-loop-helix protein. Activated AhR then binds to xenobiotic response elements (XREs) located in the regulatory regions of its target genes. XREs have the consensus sequence 5’-TNGCGTG-3’, of which the half-site, TNGC, is recognized by AhR, and the GTG motif is recognized by ARNT.¹ Metabolism (bioactivation and detoxication) of xenobiotics is one of the main biological functions of AhR target genes, such as cytochrome P450 1a1 (Cyp1a1). Because several ligands that have a high affinity to AhR are toxins and carcinogens, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene, AhR was studied initially by focusing on its role in modifying the toxicity of these chemicals. In addition to this well-established aspect of AhR centered upon the metabolism of xenobiotic compounds, several lines of evidence have revealed physiological functions of AhR where it was reported that AhR plays important roles in maintenance of barriers in multiple organs such as the placenta, lung, gastrointestinal tract, and liver, immunomodulation, and tissue development.^2–4 Further, recent studies report potential crosstalk between AhR and other signaling pathways,^5,6 such as HIF signaling,⁷ estrogen receptor,^8,9 Wnt signaling,¹⁰ and Nrf2 signaling,^11,12 suggesting a wider range of physiological inputs from AhR. The gut harbors a dynamic interface between the host organ (i.e., the intestinal epithelial layer) and physiological stimuli, such as exposure to food-derived compounds, microbiota metabolites, and many other metabolites produced from various metabolic pathways that are potentially capable of binding to the AhR. This unique biological setting raises questions about the physiological roles of AhR specifically in the gut. Recent studies demonstrated the protective effects of AhR activation against disturbance of gut health, where underlying mechanisms appear to be related to gut barrier integrity,^13–15 and regulation of inflammatory reactions.^16,17 In addition, a few studies suggest that the AhR signaling pathway has an association with cell differentiation and stem-cell biology in the intestine^13,18,19 However, the detailed molecular mechanism by which AhR mediates the regulatory system of intestinal cell differentiation remains largely unknown.

All cell types in the intestinal epithelium are derived from self-renewing stem cells that reside at the base of the intestinal crypt unit. The genetic program orchestrated by a complex transcriptional network controls the cell differentiation process, where the earliest cell fate determination occurs in the progenitor cells regulated by the Notch signaling pathway. The intestinal stem/progenitor cells are committed either to a secretory or absorptive lineage based on the expression of key Notch downstream effectors: atonal homolog 1 (ATOH1, also termed MATH1 in mice) or Hes family bHLH transcription factor 1 (HES1).²⁰ MATH1 is a basic helix-loop-helix transcription factor playing an important role in cell fate determination in multiple tissues including neurons, auditory hair cells in inner cells, and secretory cells in the gut. Particularly, in the context of the intestine, notable phenotypes observed in Math1-deficient mice exhibit intestines populated by enterocytes with loss of all secretory cells,²¹ highlighting the role of MATH1 as a gatekeeper regulating the differentiation balance between secretory or absorptive lineages. Along with its biological aspect, the molecular characterization of MATH1 as one of the basic helix-loop-helix transcription factors has been studied. A comprehensive Math1 targetome approach employing ChIP- and RNA-sequence analysis revealed that MATH1 binds to a 10-nucleotide palindrome with an E-Box at its core, regulating genes involved in migration, cell adhesion, and metabolism in the postnatal cerebellum.²² A similar approach particularly focusing on the intestine demonstrated additional DNA binding motifs of MATH1, including but not limited to an E-box motif and several other transcription factor binding sites, such as E2A, HEB, RUNX1, and HLTF,²³ of which E2A is known to interact with the E-box elements as a heterodimer with MATH1.²⁴ Regarding the role of MATH1 as a cell fate regulator in the intestine, previous studies reported that MATH1 binds to the core promoter regions of identified MATH1 target genes, such as Sox9, Gfi1, and Spdef that are known to regulate secretory cell differentiation.^23,25–27 These studies suggest that as one of the downstream effectors of the Notch cascade, MATH1 works as a “secondary” transcription factor that induces effector genes over the time-course of cell differentiation.

Focusing on the upstream regulatory mechanism of Math1, we previously reported that the transcription factor NF-E2 p45-related factor 2; encoded by Nfe2l2 (Nrf2), which plays a critical role in regulating the expression of a battery of detoxifying and antioxidant defense genes, also negatively regulates the expression of Math1. This NRF2-Math1 axis, found in progenitor cells in the small intestine, contributes to enterocyte differentiation.²⁸ In this study, we demonstrated that AhR binds to XREs in the regulatory region of Math1 and regulates Math1 expression. The AhR-Math1 transcriptional axis was further investigated in the murine small intestine, where the HES1-Math1 axis was suggested to coexist with this putative AhR-Math1 transcriptional axis within the regulatory region of Math1. Under the condition in which the HES1-Math1 axis was suppressed, an enhanced population of Math1⁺ progenitor cells was observed in intestinal organoids and small intestine upon ligand activation of AhR. Further, the secretory commitment of those Math1⁺ progenitor cells was suggested from organoids harboring an increased number of goblet cells. Our findings unveiled a novel crosstalk between the Notch signaling cascade and AhR, further highlighting the potential of targeting AhR as a part of preventive and/or therapeutic strategies for optimization of gut health by modifying homeostasis of cell differentiation via Math1 transcriptional regulation.

RESULTS

AhR binds to XREs in the regulatory region of Math1 in intestinal crypt cells

The proximal promoter region of atonal homolog 1 (ATOH1) gene in humans and Math1 in mice is highly conserved between the two species. Both possess two putative XREs (Fig. 1A). Math1 is an important gene regulating intestinal cell fate determination in the small intestine as a downstream effector of the Notch signaling pathway.²⁹ Thus, it was hypothesized that the potential transcriptional regulation of Math1 by AhR may affect the cell fate regulatory cascade in the small intestine. As an initial approach, the binding activity of AhR to XREs in the regulatory region of Math1 in intact live cells was determined using intestinal cells. The anti-AhR antibody, whose specificity and selectivity were confirmed in cells treated with AhR ligands, was used for a chromatin immunoprecipitation (ChIP) assay. To directly study the potential AhR-Math1 axis in the context of cell differentiation signaling in the small intestine, purified crypt cells derived from β-NF-treated mice were used. Wild-type mice were treated with one dose of β-NF (40 mg/kg BW), in which the effect of AhR activation in the small intestine was confirmed by the elevated expression of Cyp1a1, a prototypical AhR target gene (Fig. 1B). DNA extracted from the input and the immunoprecipitation fractions were amplified using the primers corresponding to the XRE-containing region of the Math1 regulatory site. The XRE in the Cyp1a1 promoter region was examined as a positive control. As shown in Fig. 1C, the crypt cells derived from the β-NF-treated mice showed significantly higher intensity bands corresponding to Math1 XRE and Cyp1a1 XRE compared to the crypt cells from vehicle-treated mice, demonstrating the binding activity of AhR to the XREs in the regulatory region of Math1 in the crypt cells.

FIG 1.

AhR binds to XREs in the regulatory region of Math1. (A) The proximal promoter sequence of the mouse Math1 and human ATOH1 gene, featuring XREs (xenobiotic response elements, highlighted in orange), an N-box (highlighted in blue), and a translation start site (highlighted in red). For ATOH1, the 5′ flanking region is shown. (B) Schema of treatment for mice (top panel), and Cyp1a1 mRNA expression in intestinal mucosal tissue (bottom panel). Wild-type mice were treated with one dose of β-NF (40 mg/kg BW, i.p.) and intestinal mucosal tissues were collected 5 h later. Gene expression was normalized by Actb, and the expression level in the vehicle control was set as 1. *P < 0.05 using Student’s t test. (C) ChIP assay using single intestinal crypt cells prepared from the isolated crypts of β-NF treated-mice. Control mouse genome, chromatin immunoprecipitants with IgG, and AhR were used for PCR amplification of the XREs in the Math1 regulatory region (upper gel), and in the Cyp1a1 regulatory region (lower gel). The binding of AhR to the XRE in the regulatory region of Cyp1a1 was used as a positive control. IgG was used as the negative control for each assay. The relative intensities of bands are shown in the bottom panels. The biological replicates (n = 3) per treatment are shown. Values are mean ± S.D. *P < 0.05 using Student’s t test.

XREs in the regulatory region of Math1 contribute to the regulation of Math1 expression by ligand-mediated AhR activation

To examine transcriptional regulation of Math1 by AhR, the promoter region of Math1 (∼ 400 bp upstream from translation initiation codon) was isolated and ligated into the luciferase reporter pGL3 basic vector (p-266 Math1-Luc)²⁸ as shown in Fig. 2A. The two XREs found in the proximal promoter region were named I-XRE and II-XRE, respectively in this study. For the studies of molecular regulation, Hepa 1–6 hepatoma cells and HEK293 human embryonic kidney cells were selected on the basis of previous studies using these particular cell lines exhibiting high levels of AhR expression coupled with ease of transfection for studying the regulatory mechanisms of gene transcription in mediating AhR signaling. First, Hepa 1–6 cells were transfected with a p-266 Math1-Luc construct, and luciferase activity was compared between vehicle treatment and treatment with four AhR ligands: 3-methylcholanthrene (3MC), β-naphthoflavone (β-NF), indirubin (Ind), or kynurenine (Kyn). The luciferase activity was significantly higher in the cells treated with low concentrations of 3-MC, Ind, or Kyn compared with vehicle, and the treatment with β-NF showed similarly an increasing trend of luciferase activity (Fig. 2B). A reporter construct harboring point mutations in both I-XRE and II-XRE (p-266 I, II-XRE mut Math1-Luc) was prepared for further study, resulting in abrogation of elevated luciferase activity following administration of 3-MC, β-NF, Ind, or Kyn (Fig. 2C). Given our previous findings demonstrating NRF2-mediated transcriptional regulation of Math1,²⁸ the possibility of intersectional transcriptional activity of Math1 by antioxidant response elements (AREs) and XREs was examined using a ARE-mutated construct harboring the point mutation in two AREs found in the proximal promoter region of Math1 (p-266 I, II-ARE mut Math1-Luc) (Fig. 2C). The cells transfected with p-266 I, II-ARE mut Math1-Luc showed significantly elevated luciferase activity following treatment with the examined AhR ligands, wherein induction levels were comparable to that observed with p-266 Math1-Luc (Fig. 2B), indicating that the AhR-Math1 transcriptional regulation was independent of AREs.

FIG 2.

AhR regulates Math1 expression in a XRE dependent manner. (A) Diagram of the p-226 Math1-Luc reporter construct. (B) The relative luciferase activities of Hepa 1–6 cells transfected with p-226 Math1-Luc reporter. The cells were treated with vehicle, or the AhR inducers 3-MC (3-methylcholanthrene, 50 nM), β-NF (β-naphthoflavone, 200 nM), Ind (indirubin, 250 nM), or Kyn (L-kynurenine, 480 nM) for 24 h until assay. (C) The relative luciferase activities of Hepa 1–6 cells transfected with the XRE (xenobiotic response element) mutant reporter (p-266 I, II-XRE mut Math1-Luc), and the ARE (antioxidant response element) mutant reporter (p-266 I, II-ARE mut Math1-Luc). The two mutant reporters used in this experiment harbor point mutations in two XREs (I-XRE and II-XRE) and two AREs (I-ARE and II-ARE), respectively. (D) The relative luciferase activities of Hepa 1–6 cells transfected with p-226 Math1-Luc reporter, and a battery of reporter constructs harboring a point mutation in II-XRE (p-266 II-XRE mut Math1-Luc), a point mutation in I-XRE (p-266 I-XRE mut Math1-Luc), and point mutations in I-XRE and II-XRE (p-266 I, II-XRE mut Math1-Luc). The cells were treated with vehicle or 3-MC (20 nM) for 24 h before harvest. The luciferase activities were normalized by the Renilla luciferase activity from a co-transfected reporter vector throughout, and the activity values in the vehicle-treated cells were set at 1.0. (E) Western blot analysis of MATH1 and Lamin B expression in lysates of whole nuclei prepared from Hepa 1–6 cells treated with vehicle, 3-MC (100 nM), or β-NF (200 nM) for 20 h. The normalized expression levels of MATH1 relative to Lamin B in the vehicle-treated cells were set at 1.0. A total of three independent experiments were performed, of which two of experiments are presented in the blotting image. *P < 0.05 using one-way ANOVA followed by Tukey's test.

Next, to further characterize the contribution of the two XREs to Math1 transcriptional regulation, the mutant constructs having a single point mutation in either I -XRE or II-XRE (p-266 I-XRE mut Math1-Luc and p-266 II-XRE mut Math1-Luc, respectively) were generated (Fig. 2D). Then 3-MC evoked attenuated elevation of luciferase activity in the cells transfected with p-266 I-XRE mut Math1-Luc or p-266 II-XRE mut Math1-Luc, whilst the p-266 I, II-XRE mut Math1-Luc construct completely blunted the response seen in the positive control of p-266 Math1-Luc. In addition to transcriptional regulation of Math1 by ligand-mediated AhR activation, protein expression of MATH1 was examined following administration of the AhR ligands. Hepa 1–6 cells treated with 3-MC or β-NF increased protein expression of MATH1 in the nuclear extracts compared to the vehicle-treated cells (Fig. 2E). Collectively, these data demonstrated that ligand-mediated AhR activation induced XRE-dependent transcriptional regulation of Math1 and subsequent elevation of MATH1 protein expression. Further, both I-XRE and II-XRE in the promoter region of Math1 are responsible for the AhR-Math1 transcriptional axis.

ChIP assays as done in Fig. 1C with intestinal crypt cells were also conducted using Hepa 1–6 cells treated with two AhR ligands, 3-MC and β-NF. They showed binding activity of AhR to the XREs in the Math1 regulatory region and the XREs in the Cyp1a1 regulatory region, supporting the potential for an AhR-Math1 transcriptional axis. To further determine transcriptional activity of AhR on Math1, an electrophoretic mobility shift assay (EMSA) was performed by employing the cells harboring the overexpression of a heterodimeric transcription factor formed by AhR and ARNT. Using a 4xXRE-Luc reporter construct (p4xXRE-tk-Luc), it was confirmed that co-transfection of the AhR and ARNT expression vectors achieved significantly higher luciferase activities in the ligand-activated conditions compared to the control cells (Fig. 3A). There is no statistical difference in the basal state of luciferase activity between the cells having forced expression of AhR and ARNT, and the mock control cells. Using these validated expression vectors, the mixture of nuclear extracts prepared from the cells having forced expression of AhR and ARNT and the probes containing the I-XRE or II-XRE site of the Math1 promoter region were examined, respectively (Fig. 3B). A band indicating a complex motif of protein and DNA was observed in both assays for I-XRE and II-XRE (Fig. 3B, lane 2). These bands completely disappeared when an excess amount of unlabeled probes (competitors) for either Math1 I-XRE or Math1 II-XRE were added (Fig. 3B, lanes 3 and 4). Further, when the probe containing the XRE of the Cyp1a1 promoter region was used as a competitor, the band of the complex motif became significantly weakened, which did not occur when the mutated probe for XRE was added as a competitor (Fig. 3B, lane 5 and 6). To specifically examine protein binding to either I-XRE or II-XRE, an EMSA supershift assay was performed. In assays for I-XRE and II-XRE, the bands of the complex motif of protein and DNA (Fig. 3C, lane 2) were observed as a shifted band when the anti-AhR antibody was added (Fig. 3C, lane 4). These data indicate AhR binds to both I-XRE and II-XRE in the promoter region of Math1. Taken together, it was observed that ligand-mediated AhR activation regulated transcription of Math1 through its direct binding to the XREs in the promoter region of Math1, subsequently elevating MATH1 protein expression. It was not possible to detect Math1 transcript levels in the Hepa 1–6 cells we used in the in vitro experiment, possibly due to a short half-life of Math1 mRNA. It has been reported that degradation of Math1 mRNA by another bHLH transcription factor, NEUROG1 occurred within 1 h. In that setting the rapid suppression of Math1 by NEUROG1 is an important mechanism to prevent hair cell development during neurogenesis, and degradation of Math1 mRNA was indicated to be an underlying mechanism.³⁰

FIG 3.

AhR binds to XREs in the regulatory region of Math1. (A) Functional confirmation of the AhR expression vector (pCMV AhR) and the Arnt expression vector (pEF Arnt) using p4xXRE-tk-Luc reporter constructs. Hepa 1–6 cells were co-transfected with pCMV AhR and pEF Arnt, or their mock vectors, and then treated with vehicle, 3-MC (2 µM), or β-NF (10 µM) for 24 h before assay. The concentrations of the AhR ligands used in this specific assay were matched to a previous publication.^{58 #}P < 0.05 vs vehicle-treated mock control, ^$P < 0.05 vs vehicle-treated pCMV AhR + pEF Arnt, and *P < 0.05 for indicated comparison using one-way ANOVA. Three independent experiments were performed for each assay. (B) Standard EMSA assay to examine binding of AhR to I-XRE (left panel) and II-XRE (right panel) in the regulatory region of Math1. Nuclear extracts prepared from HEK293 cells having forced expression of AhR and ARNT were used. For the competition experiments, a 100-fold molar excess amount of the unlabeled probes were used. Lane 1: negative control (no nuclear protein); Lane 2: control (nuclear protein extracted from HEK293 cells); Lane 3: nuclear protein plus unlabeled probe for Math1 I-XRE; Lane 4: nuclear protein plus unlabeled probe for Math1 II-XRE; Lane 5: nuclear protein plus unlabeled probe for the XRE in the regulatory region of Cyp1a1; Lane 6: nuclear protein plus unlabeled mutant probe for XRE. The arrowheads indicate the DNA-protein complexes. (C) The EMSA supershift assay for I-XRE (left panel) and II-XRE (right panel) using Hepa 1–6 cells treated with 3-MC (100 nM). Lane 1: negative control (no nuclear protein); Lane 2: control (nuclear protein from Hepa 1–6 cells); Lane 3: nuclear protein plus antibody against IgG; Lane 4: nuclear protein plus antibody against AhR. The white arrowheads indicate the shifted bands. The black arrowheads indicate the DNA-protein complexes. All EMSA experiments were repeated three times utilizing separate nuclear extracts from cells. Values are mean ± SD. COMP; competitors.

AhR activation by low concentration of β-NF does not affect cell differentiation in intestinal organoids

The expression of MATH1 in the intestinal crypt cells initiates their differentiation toward secretory cell lineages. To investigate how the AhR-Math1 axis, in which a transcriptional linkage was demonstrated in cell lines and intestinal crypt cells, affects cell differentiation in the small intestine, mouse intestinal organoids were employed. Organoids derived from wild-type mice were treated with β-NF (5 nM or 50 nM) for 2 days as shown in Fig. 4A. To investigate the physiological roles of AhR, lower concentrations of β-NF were used compared to µM ones often used in in vitro studies,^31–33 wherein the role of AhR in pathological settings (i.e., intestinal carcinogenesis, radiation-induced intestinal injury) were pursed. The activation of AhR signaling in the cultured organoids by β-NF treatment was confirmed by measuring mRNA expression of Cyp1a1 (Fig. 4B). Gene expression levels of cell markers including stem cell markers (Lgr5, Olmf4), secretory cell markers (Muc2, Chga, Lyz), and absorptive cell marker (Apoa1) did not show any changes between the β-NF-treated organoids and the controls (Fig. 4C). The histological evaluation of goblet cells, which is one of the cell types in the secretory cell lineage, was performed using Alcian blue staining, showing that β-NF treatment did not change the number of goblet cells in the organoids (Fig. 4D and E).

FIG 4.

AhR activation by low concentration of β-NF does not affect cell differentiation in intestinal organoids. (A) Schema for treatment design. The cultured organoids were treated with β-NF (β-naphthoflavone, 5 nM and 50 nM) or vehicle for two days starting from culture day 5 and collected on culture day 7. (B) Cyp1a1 mRNA expression in the organoids treated with vehicle, 5 nM β-NF, or 50 nM β-NF. (C) Gene expression of stem cell markers (Lgr5, Olmf4), secretory cell markers (Muc2, Chga, Lyz), and absorptive cell marker (Apoa1) in the organoids. Gene expression was normalized by Actb, and the expression level in the vehicle control was set as 1. Three independent experiments were performed. (D) Alcian blue staining for the organoids treated with vehicle or β-NF. White arrowheads indicate Alcian blue-stained goblet cells in the organoids. Bar, 50 µm. (E) Left panel: The frequency of the organoids classified by the number of goblet cells normalized by the cross-sectional area of the corresponding organoid. Classification of values were distributed as 0–1, 1–5, 5–10, 10–15, 15–20, 20–25, and more than 25. In each experiment, 60–200 organoids in each treatment group were examined to obtain frequency values, of which the averages are represented. Four independent experiments were performed. Right panel: The frequency of organoids classified as quantification values of goblet cells less than 1. Individual frequency numbers obtained from four independent experiments are presented. Values are mean ± SD. *P < 0.05 using one-way ANOVA followed by Tukey's test. V; vehicle.

AhR activation by β-NF in the presence of γ-secretase inhibitor accelerates goblet cell differentiation in intestinal organoids

HES1 is a Notch signaling component that represses bHLH transcriptional activators including MATH1. The negative regulation of Math1 by HES1 is reported to be important for cell differentiation in inner ear hair cells and secretory cells in the small intestine.^34,35 Considering the unique sequence of the Math1 regulatory region harboring one of the XREs (II-XRE) and an HES1-binding motif (N-box) located 1 bp apart (Fig. 1A and Fig. 5A), a complex and/or competitive transcriptional regulation of Math1 by AhR and HES1 was expected. In order to directly investigate the biological contributions of the AhR-Math1 axis in the small intestine, it was necessary to interfere with the HES1-Math1 transcriptional axis. Thus, the potent inhibitor of Notch signaling, γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT) was employed before administration of β-NF as shown in Fig. 5B, where suppression of Hes1, the secondary effector of Notch signaling was targeted to inhibit the HES1-Math1 axis in the intestinal organoids. To discriminate the primary purpose of the DAPT administration (i.e., suppressing Hes1 as the subsequent outcome of Notch inhibition) from concomitant effects on cell differentiation occurring from DAPT treatment, the key comparative analysis in this study was performed between DAPT alone treatment and DAPT plus β-NF treatment. Expression of Hes1 was significantly inhibited by DAPT treatment in the organoids (Fig. 5C). It was also confirmed that a sequential administration of DAPT and β-NF adequately evoked suppression of Hes1 as well as activation of AhR signaling (Fig. 5C). The suppression level of Hes1 in a sequential treatment (Fig. 5C, middle panel) was milder than the one occurred in the organoids treated only with DAPT (Fig. 5C, left panel), which is likely due to the washout effect of DAPT during the period of β-NF treatment. Measures of gene expression markers demonstrated that both DAPT followed by vehicle and DAPT followed by β-NF treatment showed significantly lower expression of the stem cell marker Olmf4 than vehicle control, while the expression of secretory cell markers, specifically Muc2 and Chga were higher than control (Fig. 5D). Although the expression of Apoa1 did not show a clear trend, another cell marker of absorptive cell lineage, Alpi was lower in the organoids pretreated with DAPT than in the control organoids. These data indicated that DAPT treatment arrested crypt cell proliferation and caused the profound acceleration of secretory cell differentiation in the organoids. The observations are consistent with previous reports of the response of mouse small intestines to treatment with the γ-secretase inhibitor.³⁶ It was also revealed that DAPT alone and DAPT followed by β-NF treatment showed comparable effects on the expression of all examined cell markers, demonstrating that AhR activation by β-NF did not show added effects on DAPT pretreatment in the organoids with mRNA expression of secretory and absorptive cell markers.

FIG 5.

AhR activation by β-NF coupled with pharmacological suppression of the HES1-Math1 axis induces goblet cell differentiation in intestinal organoids. (A) Map of the Math1 promoter site (transcription start site (+1)) indicating the distribution of putative XRE (xenobiotic response element) motifs and N-box. (B) Schema for sequential treatment design. The cultured organoids were treated with DAPT (10 µM) or vehicle for three days starting from culture day 2, followed by β-NF (β-naphthoflavone, 5 nM and 50 nM) or vehicle treatment for two days. The samples were collected at culture day 7. (C) Hes1 mRNA expression in the organoids treated with DAPT alone (left panel), and DAPT followed by β-NF treatment (middle panel), and Cyp1a1 mRNA expression in the organoid treated with DAPT followed by β-NF (right panel). For DAPT-alone treatment, the organoids were treated with DAPT for three days and collected at culture day 4. Student’s t test was performed for Hes1 mRNA expression (left panel). Three or five independent experiments were performed. The gray and blue bars correspond to the organoids exposed to vehicle pretreatment or DAPT pretreatment, respectively. (D) Gene expression of stem cell markers (Lgr5, Olmf4), secretory cell markers (Muc2, Chga, Lyz), and absorptive cell markers (Apoa1, Alpi) in the organoids. Gene expression was normalized by Actb, and the expression level in the vehicle control was set as 1. Five independent experiments were performed. (E) Alcian blue staining for the organoids treated with vehicle, DAPT followed by vehicle, and DAPT followed by β-NF (5 nM and 50 nM). White arrowheads indicate Alcian blue-stained goblet cells in the organoids. Bar, 50 µm. (F) Left panel: The frequency of organoids classified by the number of goblet cells normalized by the cross-sectional area of the corresponding organoid. Classification of values were distributed as 0–1, 1–5, 5–10, 10–15, 15–20, 20–25, and more than 25. In each experiment, 60–200 organoids in each treatment group were examined to obtain frequency values, of which the averages are represented. Four independent experiments were performed. Right panels: The frequency of organoids classified as quantification values of goblet cells less than 1 or more than 15. The individual frequency numbers obtained from the four independent experiments are presented. One-way ANOVA was performed and P values with statistical significances are shown. Values are mean ± SD. *P < 0.05 using one-way ANOVA followed by Tukey's test unless otherwise noted. V; vehicle.

Of note, suppression of the Notch signaling pathway by the γ-secretase inhibitor induced unusual secretory cells that were co-stained by both goblet cell and Paneth cell markers,³⁷ which may partially explain the large variation in mRNA expression of the cell markers for these cell types in the organoids. The principal purpose of DAPT pretreatment in this study is to inhibit the HES1-Math1 transcriptional axis by suppressing Hes1 expression before administration of β-NF. Thus, the effects of DAPT pretreatment likely causing irregular secretory cells in the organoids were excluded from our evaluation. In addition, given previous studies suggesting an association between modulation of AhR signaling and differentiation of goblet cells in the small intestine,^13,19 we determined the effects on goblet cell differentiation by enumerating Alcian blue-stained cells in the organoids treated with β-NF coupled with DAPT. As shown in Fig. 5E, DAPT treatment alone showed increased numbers of goblet cells in organoids compared to vehicle control. Crucially, the organoids harboring greater numbers of goblet cells were more frequently observed in the DAPT followed by β-NF (50 nM) treatment group as compared to DAPT alone. This difference was statistically significant (Fig. 5E and F, right panel, P value = 0.045); on the contrary, the organoids with fewer numbers of goblet cells showed an increased frequency in the DAPT alone treatment compared to the DAPT and β-NF (50 nM) treatment (Fig. 5E and F, middle panel). Although the added effect on goblet cell numbers following DAPT plus β-NF treatment was smaller when 5 nM β-NF was used, the similar trend was confirmed. Together, these data indicate that under the suppression of the HES1-Math1 axis achieved by DAPT administration, β-NF-mediated activation of AhR increases the number of Alcian blue-stained goblet cells in the intestinal organoids.

DAPT plus β-NF treatment accelerates goblet cell differentiation in intestinal organoids in an AhR-dependent manner

To interrogate further the effects of AhR on goblet cell differentiation in the organoids by DAPT plus β-NF treatment, AhR^F/F::VilCre mice were used. The expression level of Cyp1a1 in the intestinal mucosal tissues was lower in AhR^F/F::VilCre mice than in AhR^F/F mice, verifying the functional suppression of AhR signaling in the small intestine of AhR^F/F::VilCre mice (Fig. 6A). The organoids derived from AhR^F/F::VilCre mice were treated with DAPT followed by β-NF in the same manner as the treatment performed for wild-type organoids (Fig. 6B). It was also confirmed that the concentrations of β-NF used in this study (5 nM or 50 nM) did not elevate the expression of Cyp1a1 in the AhR^F/F::VilCre organoids (Fig. 6C) unlike in wild-type mice. In addition, it was confirmed that both DAPT-alone treatment and DAPT plus β-NF treatment suppressed Hes1 expression in the AhR^F/F::VilCre organoids (Fig. 6C), wherein the effects were comparable with the those observed in the wild-type organoids. DAPT-alone treatment increased the number of Alcian blue-stained goblet cells in the AhR^F/F::VilCre organoids to a similar degree with wild-type organoids (Fig. 6D and E). The notable point is that the added effects on goblet cell numbers observed in the wild-type organoids treated by DAPT plus β-NF was abolished in the AhR^F/F::VilCre organoids. Taken together, these data demonstrated that AhR activation coupled with Hes1 suppression by DAPT treatment increased the number of goblet cells in the organoids in an AhR-dependent manner.

FIG 6.

Genetic knockout of AhR abolishes β-NF-mediated effects on goblet cells in intestinal organoids. (A) Cyp1a1 mRNA expression in the intestinal mucosal tissues of AhR^F/F (n = 6) and AhR^F/F::VilCre (n = 7) mice. Gene expression was normalized by Actb, and the expression level in AhR^F/F was set as 1. *P < 0.05 using Student’s t test. (B) Schema for treatment design. The cultured organoids derived from AhR^F/F::VilCre mice were treated with DAPT (10 µM) or vehicle for three days starting from culture day 2, followed by β-NF (β-naphthoflavone, 5 nM or 50 nM) or vehicle for two days. Samples were collected at culture day 7. (C) Cyp1a1 mRNA expression in the AhR^F/F::VilCre organoids treated with DAPT followed by β-NF (left panel), and Hes1 mRNA expression in the organoids treated by DAPT alone (middle panel) and DAPT followed by β-NF (right panel). For DAPT alone, the organoids were treated with DAPT for three days and collected at culture day 4. Gene expression was normalized by Actb, and the expression level in the vehicle control was set as 1. Student’s t test was performed for Hes1 mRNA (middle panel). Four independent experiments were performed. (D) Alcian blue staining for the AhR^F/F::VilCre organoids treated with vehicle, DAPT followed by vehicle, and DAPT followed by β-NF (5 nM or 50 nM). White arrowheads indicate Alcian blue-stained goblet cells in the organoids. Bar, 50 µm. (E) Left panel: The frequency of the organoids classified by the number of goblet cells normalized by the cross-sectional area of the corresponding organoid. Classification of values were distributed as 0–1, 1–5, 5–10, 10–15, 15–20, 20–25, and more than 25. In each experiment, 60–200 organoids in each treatment group were examined to obtain frequency values, of which the averages are presented. Three independent experiments were performed. Right panel: The frequency of organoids classified as quantification values of goblet cells more than 15. The individual frequency numbers obtained from the three independent experiments are presented. Values are mean ± SD. *P < 0.05 using one-way ANOVA followed by Tukey's test. V; vehicle.

The consecutive treatment with β-NF and γ-secretase inhibitor increases Math1⁺ progenitor cells in intestinal organoids

MATH1 is an important player regulating a binary cell fate decision between secretory and absorptive cell lineages by directing the commitment of Notch⁺/Hes1⁺ absorptive progenitor cells and Math1⁺ secretory progenitor cells.³⁸ Given that AhR-mediated acceleration of goblet cell differentiation required suppression of the HES1-Math1 axis in the organoids, Math1⁺ progenitor cells, that synchronously do not express HES1, were hypothesized to be the potential target cells utilizing the AhR-Math1 axis to modify secretory cell fate in the organoids. To approach this hypothesis, Math1-GFP reporter mice were used. As reported previously^23,39 it was confirmed that Math1-GFP is expressed in some of the crypt cells as well as in the cells located in the villi of the small intestines (Fig. S1A). Using the intestinal organoids, it was further validated that Math1-GFP expression was co-localized with the expression of markers for three types of secretory cells (i.e., goblet cells, enteroendocrine cells, Paneth cells) (Fig. S1B). It was also shown that MUC2-stained goblet cells included a certain population of GFP-none expressing cells (approximately 30–40%) in the small intestine, suggesting the expression pattern of GFP, particularly in the matured secretory cells of this reporter mouse, is reflected by the persistence of GFP protein and not accurately useful for lineage tracing for Math1. However, as with previous reports of fate mapping using well-established Math1-reporter mice,^29,39 our observations confirm that all types of secretory cells arise from common secretory progenitor cells that express Math1. To distinguish between Math1⁺ progenitor cells and Math1⁺ mature secretory cells in Math1-GFP reporter mice, the Math1-GFP and Ki67 double positive cells were defined as Math1⁺ secretory progenitor cells, as studied previously.²⁹ The totality of Math1-GFP positive cells (green circle in Fig. 7A) include all types of mature secretory cells and secretory progenitor cells, while the totality of Ki67 positive cells (red circle in Fig. 7A) include intestinal stem cells and two types of progenitor cells (i.e., Math1⁺ secretory progenitor cells, Notch⁺/Hes1⁺ absorptive progenitor cells). Thus, the Math1-GFP and Ki67 double-positive cells are considered to be proliferative Math1-expressing cells that will be committed to the secretory cell lineage. The organoids derived from Math1-GFP mice were treated with vehicle or β-NF (50 nM) (Fig. 7B, upper panels), or DAPT pretreatment followed by vehicle or β-NF (50 nM) (Fig. 7B, lower panels) in the same manner as shown in Fig. 5B. The organoids pretreated with DAPT showed an increasing trend in the number of Math1-GFP positive cells and a decreasing trend in Ki67 positive cells compared to the organoids that were not administrated DAPT (Fig. 7C to F). These observations showing a conversion of crypt proliferative cells to secretory types of cells were consistent with the results of cell marker expression shown in the wild-type organoids (Fig. 5D). The organoids treated with β-NF alone did not show any changes in the numbers of total and single Math1-GFP-positive cells (Fig. 7C) and in the number of total and single Ki67-positive cells (Fig. 7E). A sequential treatment with DAPT and β-NF did not show any alteration in numbers of total and single Math1-GFP-positive cells (Fig. 7D). The organoids that have a greater number of total Ki67-positive cells (i.e., a number of Ki67-positive/Math1-GFP-positive cells and Ki67-positive/Math1-GFP-negative cells) were more frequently observed in the organoids treated with DAPT plus β-NF compared to DAPT-alone treatment (a statistically significant interaction, [P value = 0.023] between the treatment condition and total Ki67-positive cell number on frequency value was shown by two-way ANOVA), whereas the number of single Ki67-positive cells (i.e., a number of Ki67-positive/Math1-GFP-negative cells) did not show a statistical difference between the two treatment groups (Fig. 7F). Most importantly, Math1-GFP and Ki67 double-positive cell numbers were notably elevated by the sequential treatment compared to DAPT alone (Fig. 7H, a statistically significant interaction (P value = 0.0002) between the treatment condition and double-positive cell number on frequency value was shown by two-way ANOVA), which was not observed between the organoids treated with vehicle and β-NF alone (Fig. 7G). In summary, DAPT-alone treatment forced cells to exit the cell cycle to become post-mitotic cells, whereas AhR activation by β-NF in the presence of DAPT showed an increased number of Math1⁺ progenitor cells in the organoids. Given the increased number of Alcian blue-stained cells in the organoids treated by DAPT plus β-NF (Fig. 5E and F), it is plausible to assume that the Math1⁺ progenitor cell pool enhanced by DAPT plus β-NF treatment leads to the promotion of secretory commitment, wherein goblet cells are likely to be one of the potential secretory cell lineages that can be affected by AhR activation.

FIG 7.

AhR activation by β-NF in the presence of γ-secretase inhibitor increases Math1⁺ progenitor cells in intestinal organoids. (A) Math1-GFP positive cells (green circle) include all types of mature secretory cells and secretory progenitor cells. Ki67 positive cells (red circle) include stem cells and two types of progenitor cells (i.e., secretory progenitor cells, absorptive progenitor cells). Thus, the Math1-GFP and Ki67 double-positive cells indicated by the arrow are considered to be proliferative Math1-GFP-positive cells (secretory progenitor cells). (B) Immunofluorescent images for the intestinal organoids derived from Math1-GFP mice (Math1-GFP^+/+). The organoids were treated with vehicle, β-NF (β-naphthoflavone, 50 nM), DAPT (10 µM) followed by vehicle, and DAPT (10 µM) followed by β-NF (50 nM). Green, GFP; Red, Ki67; Blue, DAPI. Yellow arrows indicate the double-stained cells by GFP and Ki67. Bar, 30 µm. (C and D) The frequency of the organoids classified by the number of total Math1-GFP positive cells (Math1-GFP positive/Ki67 positive cells and Math1-GFP-positive/Ki67-negative) and single Math1-GFP-positive cells (Math1-GFP positive/Ki67-negative). The number of Math1-GFP positive cells was counted in each organoid and normalized by total cell number (DAPI-positive cell number). The frequency of evaluation values (less than 0.1 or more than 0.1) was compared between the organoids treated with vehicle and β-NF (C) and DAPT followed by vehicle and DAPT followed by β-NF (D). (E and F) The frequency of the organoids classified by the number of total Ki67 positive cells (Ki67-positive/Math1-GFP-positive and Ki67-positive/Math1-GFP negative), and single Ki67-positive cells (Ki67-positive/Math1-GFP-negative). The number of Ki67-positive cells was counted in each organoid and normalized by total cell number. The frequency of evaluation values (less than 0.3 or less than 0.5, and more than 0.3 or more than 0.5) was compared between the organoids treated with vehicle and β-NF (E) and DAPT followed by vehicle and DAPT followed by β-NF (F). (G and H) The frequency of the organoids classified by the number of Math1-GFP and Ki67 double-positive cells. The number of double-positive cells was counted in each organoid and normalized by the total cell number. The evaluation values were classified as 0–0.01, 0.01–0.03, 0.03–0.05, 0.05–0.07, 0.07–0.09, 0.09–0.11, and more than 0.11. The frequency of evaluation values (less than 0.01 and more than 0.01) was compared between the organoids treated with vehicle and β-NF (G, left panel) and DAPT followed by vehicle and DAPT followed by β-NF (H, left panel). The distributions of values are presented in the right panels. At least 25 organoids in each treatment group were examined in each experiment; four or five individual experiments were performed. Values are mean ± SD. Two-way ANOVA was performed to analyze the data, where one factor was the treatment condition (vehicle and β-NF, or DAPT plus vehicle and DAPT plus β-NF), and the other factor was the indicated cell number (GFP-positive cell number, Ki67-positive cell number, or GFP and Ki67 double-positive cell number). The main effect of each factor was tested as well as the interaction within both factors.

The sequential treatment by γ-secretase inhibitor and β-NF increases Math1⁺ progenitor cells in mouse small intestine

To further support biological insight into the AhR-Math1 axis in the cell fate regulatory mechanism in the small intestine, an in vivo experiment was performed. Our observations in the intestinal organoids signified that the AhR-Math1 axis in the biological setting appears to coexist with the Hes1-Math1 transcriptional axis. To dissect the singular role of the AhR-Math1 molecular axis, the sequential treatment design was applied for the in vivo experiment as well, wherein γ-secretase inhibitor-alone treatment and γ-secretase inhibitor followed by β-NF were compared to decipher AhR-mediated effects. The γ-secretase inhibitor, dibenzazepine (DBZ, (S,S)-2-[2-(3,5-difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide) is commonly used for in vivo experiments. As shown in Fig. 8A, Math1-GFP reporter mice were treated with one dose of DBZ (30 µmol/kg BW) followed by three doses of β-NF (40 mg/kg BW). It was confirmed that the single dose of DBZ sufficiently suppressed Hes1 mRNA expression in the intestinal crypts (Fig. 8B), which corresponded with a previous observation shown in Hes1 reporter mice.⁴⁰ The sequential treatment did not cause any changes in body weight (Fig. 8C), verifying no systemic toxicity during this short-term experimental period. To determine the effects of AhR activation on progenitor cells, the crypt epithelium was investigated specifically. It was shown that there were no alterations in the number of total and single Math1-GFP-positive cells and Ki67-positive cells in the intestinal crypts between DBZ plus vehicle and DBZ plus β-NF treatment (Fig. 8D and E). Notably, the sequential treatment showed a significant increase in Math1-GFP and Ki67 double-positive cell numbers in the crypts (Fig. 8D and E). Given the increased number of Alcian blue-positive goblet cells in the organoids treated with DAPT plus β-NF (Fig. 5E and F), goblet cells in the villi in the small intestine of the Math1-GFP mice were also evaluated, showing an upward trend, but no significant alteration of goblet cell number between DBZ plus β-NF treatment and DBZ alone treatment (n = 8 for each group, P value = 0.196). In aggregate, under the condition of suppressed HES1-Math1 axis, ligand-mediated AhR activation increased the population of Math1⁺ progenitor cells in the small intestine, an observation consistent with that seen in the organoids. However, the effect of increased goblet cell numbers shown in the organoids treated by γ-secretase inhibitor plus β-NF was not observed in vivo. Likely, interplay in vivo with other signal networks or other modifying factors remain to be elucidated.

FIG 8.

Sequential treatment with β-NF and γ-secretase inhibitor increases Math1⁺ progenitor cells in mouse small intestines. (A) Schema for treatment of mice. Math1-GFP mice (Math1-GFP^+/+) were treated with one dose of DBZ (30 µmol/kg BW, i.p.) followed by three doses of β-NF (β-naphthoflavone, 40 mg/kg BW, i.p.) or vehicle treatment. The samples were collected 24 h after the last treatment. (B) Expression level of Hes1 mRNA in the isolated crypts derived from the mice administrated one dose of DBZ (30 µmol/kg BW, i.p.) (n = 3) or vehicle (n = 3). The crypts were collected 24 h after treatment. Gene expression was normalized by Actb, and the expression level in the vehicle control was set as 1. (C) Body weights of mice before (pre) and after (post) the sequential treatment. (D) The immunofluorescent images for the small intestines of Math1-GFP mice (Math1-GFP^+/+). Green, GFP; Red; Ki67; Blue, DAPI. White and yellow arrows indicate the single- and the double-stained cells, respectively, by GFP and Ki67. Bar 50 µm. (E) The number of Math1-GFP positive cells (total: Math1-GFP-positive/Ki67-positive and Math1-GFP-positive/Ki67-negative cells, single: Math1-GFP-positive/Ki67-negative cells), Ki67-positive cells (total: Ki67-positive/Math1-GFP-positive and Ki67-positive/Math1-GFP-negative cells, single: Ki67-positive/Math1-GFP-negative cells), and Math1-GFP/Ki67 double-positive cells in the intestinal crypts of the mice treated by DBZ plus vehicle (n = 9) or DBZ plus β-NF (n = 9). The cell number was normalized by total DAPI-positive cells in the crypts. At least 50 of well-oriented crypts were analyzed per section. Values are mean ± SD. *P < 0.05 using Student’s t test.

DISCUSSION

The Notch-signaling pathway is fundamental to cell fate regulation in the small intestine. Utilizing its downstream effectors, HES1 and MATH1, Notch signaling forms the genetic hierarchy regulating the balance of cell differentiation between secretory and absorptive cell lineages. In this study, we demonstrate that expression of Math1 is regulated by AhR, a ligand‑activated transcription factor originally characterized as the dioxin receptor. Our study using intestinal organoids and intestines in mice revealed a new insight into the physiological role of AhR through modifying differentiation of Math1⁺ progenitor cells by associating AhR with the Notch-signaling cascade via regulation of Math1 (Fig. 9).

FIG 9.

Schematic model for the effects of AhR activation on Math1⁺ progenitor cells. Within the Notch-signaling cascade, Math1 plays an important role in regulation of intestinal cell fate. The expression of MATH1 in progenitor cells concomitantly suppresses Hes1, leading to a secretory commitment, whereas progenitor cells that do not express MATH1 allow Hes1 expression, leading to an absorptive commitment. When the expression of Hes1 was suppressed in the intestinal cells, ligand-mediated AhR activation increased the number of Math1⁺ progenitor cells in the small intestine, indicating the contribution of the AhR-Math1 axis to cell fate modification in Math1⁺/Hes1⁻ progenitor cells. In the intestinal organoids, AhR activation coupled with Hes1 suppression also showed accelerated differentiation to goblet cells, suggesting the possible terminal cell fate of Math1⁺ progenitor cells promoted by AhR activation.

One of the notable transcriptional features of Math1 is two enhancer sites found at 3.4 kb of the 3′ enhancer of the Math1 coding region in mice.⁴¹ Based on these unique enhancers and the reported functions of Math1 studied in the developmental process of several organs, it has been suggested that the gene expression machinery of Math1 involves multiple factors based on the biological context. Several proteins, such as HES1,²⁴ CDX2,⁴² HIC1,⁴³ HNF1A,⁴⁴ (ZIC1, ⁴⁵ SOX2,^46,47 have been identified as regulatory factors of Math1 expression and their functional importance have been suggested in different organs and cell types. We previously reported that NRF2, a major regulator of the antioxidant and cellular protective system, negatively regulates Math1 through NRF2 binding to AREs located in the regulatory region of Math1, including the 3′ enhancer region.²⁸ Further, it was revealed that the NRF2-Math1 transcriptional axis found in the intestinal progenitor cells contributes to the modification of enterocyte differentiation. In the current study, we identified AhR as a novel transcription factor influencing expression of Math1. A battery of evidence obtained from the cell lines and intestinal crypt cells showed clearly a direct interaction between AhR and XREs in the regulatory region of Math1 and its association with transcriptional regulation. The biological effects of the AhR-Math1 axis were addressed using an intestinal organoid culture system, wherein the competitive transcriptional regulation of Math1 driven by two binding activities, AhR to XRE and HES1 to N-box, was expected. The proximity of the two effector response elements drove us to employ a combination treatment design by using γ-secretase inhibitors (i.e., DAPT and DBZ) to inhibit induction of the secondary effector of Notch signaling, HES1, along with β-NF as an AhR ligand in order to disaggregate the actions of these two transcription factors on Math1 expression. It was shown that under a condition of suppressed Hes1 transcripts by the γ-secretase inhibitor, ligand-mediated AhR activation increased the numbers of goblet cells as well as Math1-positive and Ki67-positive cells in the organoids. The sequential treatment by γ-secretase and β-NF also elevated the number of Math1-positive progenitor cells in the small intestine of mice.

The lateral regulatory mechanism governed by the Notch-signaling cascade has been suggested to be critical for cell fate determination in the small intestine.^29,48 The stem cells are committed to two distinct types of progenitor cells: HES1-expressing progenitor cells that differentiate into the absorptive cell lineage, and HES1-negative progenitor cells harboring concomitant expression of Math1 that differentiate into the secretory cell lineage. Within this context of Notch-mediated lateral regulation of cell differentiation in the small intestine, our findings from organoids treated with DAPT plus β-NF, as well as the mice undergoing DBZ and β-NF treatment, suggest that the target cell type of AhR activation is the Math1⁺ progenitor cells that do not express Hes1. In that setting, the AhR-Math1 axis works as an intrinsic molecular axis, leading to elevated numbers of MATH1-expressing progenitor cells in the mouse small intestine. Bearing in mind the large difference in cell populations between secretory cells and absorptive cells in the small intestine (i.e., approximately 20% vs 80%), it could be assumed that there is an uneven cell distribution between Hes1⁺ absorptive progenitor cells and Math1⁺/Hes1⁻ secretory progenitor cells. Thus, the AhR-Math1 transcriptional axis is expected to function in the smaller population of cell types of the small intestine. This skewed distribution of progenitor cells required us to dissect this transcriptional axis by forcefully suppressing Hes1 expression in the organoids and in vivo in order to mimic the setting of Hes1⁻ progenitor cells. The limitation of the sequential treatments using γ-secretase inhibitors and β-NF is that γ-secretase inhibitors themselves have a significant effect on arresting proliferative crypt cells and accelerating secretory cell differentiation. However, the comparative analysis between γ-secretase-alone treatment and γ-secretase plus β-NF treatment successfully discriminated the outcomes derived from inhibited Notch signaling alone versus inhibited Notch and induced AhR signaling to indicate the functional importance of the AhR-Math1 axis in the small intestine.

Although this observation is limited principally to the organoids, it appears that the enhanced population Math1⁺ progenitor cells evoked by AhR activation in vivo promotes secretory commitment to include goblet cells. Several luminal factors (i.e., gut microbiota, microbial metabolites, and foods) vastly affect cell composition in the small intestine.^49,50 Thus, it could be reasonably assumed that in addition to Notch-Math1 mediated cell fate regulatory mechanism, those environmental factors are important regulators in vivo, defining the terminal cell fate on Math1⁺ progenitor cells within the range of secretory cell lineages (i.e., goblet cells, Paneth cells, and enteroendocrine cells). To elucidate the cell fate trajectory of Math1⁺ progenitor cells in the context of the AhR-Math1 axis in vivo, a lineage-tracing experiment using the established Math1-reporter mice^29,39 will be required.

It has been demonstrated that Math1-expressing progenitor cells possess cell fate plasticity, contributing to the reserve stem cell function within the small intestine following tissue damage in models such as irradiation damage, colitis using dextran sodium sulfate, and tumorigenesis using azoxymethane, that all evoke accelerated regeneration of damaged tissues.^39,51 In a normal homeostatic state, it is considered that plasticity from the secretory to the stem compartment is not essential in the small intestine. The biological impact of the AhR-Math1 transcriptional axis on the latent “stemness” reported in the MATH1-expressing crypt cells in the small intestine undergoing tissue damage was not examined in the current study, but also warrants further study.

Although the association between the physiological role of AhR and cell differentiation and stem cell biology including intestinal stem cells has been suggested^52,53 the multifaceted-biological functions remain insufficiently elucidated. Han et al. demonstrated that AhR^F/F::VilCre mice enhanced stem cell and progenitor cell proliferation via suppression of the FoxM1 signaling pathway.¹⁸ Another line of study showed that AhR^F/F::VilCre mice exhibited dysregulated stem cell proliferation and impaired cell differentiation in a Citrobacter rodentium infection model and with exposure to the mutagen azoxymethane. They also indicated that activation of AhR by indole-3-carbinol restricted stem cell proliferation and maintained stem cell homeostasis in the mutagen model.⁵⁴ Our study using in vivo and intestinal organoids models demonstrated that γ-secretase inhibitor plus β-NF treatment increased Ki67-positive cells compared to γ-secretase inhibitor treatment alone, particularly Math1 and Ki67 double-positive cells. We did not detect any notable differences related to the effects on stem cells in the organoids treated with DAPT and β-NF treatment (i.e., there were no significant differences in the expression of stem cell markers (Lgr5 and Olmf4) and in the cell number of Ki67-positive/Math1-negative cells between DAPT-alone treatment and DAPT plus β-NF treatment). However, the elevated number of total Ki67-positive cells observed in the organoids treated with the combination treatment may include, at least partially, stem cells in addition to Math1-positive proliferative cells, the majority of which are considered to be secretory progenitor cells. Indeed, a very small population (0.01–2%) of Math1-positive cells are reported to express stem cell markers.^39,51 Nonetheless, our findings demonstrated that the AhR-Math1 axis contributed to increasing the Math1-expressing progenitor cell population that in turn has the ability to differentiate into secretory cells.

Although more detailed studies are required, crosstalk between AhR signaling and other signaling pathways has been reported, including Nrf2.^5,6,11,12 Our previous findings related to the NRF2-Math1 transcriptional axis and the observations of the current study on the AhR-Math1 axis, coupled with signaling crosstalk between Nrf2 and AhR, drives an expectation for their roles in fine-tuning cell fate regulation mediated by Math1 in the small intestine. AhR and Nrf2, which utilize phytochemicals and microbial metabolites as agonist ligands and inducers, respectively, may regulate Math1 in an oppositional manner in response to dietary and microbiome changes to modify the cell differentiation balance between absorptive and secretory cell lineages. This potential signaling network centered on Math1 and its biological importance in the small intestine needs to be further corroborated by additional investigations employing appropriate in vivo models. An additional point of interest is the possibility of targeting the AhR-Math1 axis as a modifier of secretory cell differentiation, particularly goblet cell differentiation, whose functions are important to maintain intestinal mucosal homeostasis.⁵⁵ Although it is not clear whether underlying mechanisms are directly related to AhR signaling, the use of natural AhR ligands such as dietary components and microbiota-derived tryptophan metabolites as potential therapeutic modalities for a wide range of diseases has been attracting interest.^56,57 Further study of the AhR-Math1 axis in the small intestine will provide a deeper insight into its physiological roles, which may, in turn, provide a novel interventional and/or therapeutic strategy for optimizing intestinal homeostasis and possibly for regeneration of the intestinal epithelium after surgery or injury.

MATERIALS AND METHODS

Cell culture

Hepa 1–6 cells were purchased from ATCC, and HEK293 cells were kindly provided by Dr Sawa (Johns Hopkins University School of Medicine, USA). Short tandem repeat (STR) analysis for both cell lines verified cell line authentications (ATCC). Both cells were cultured in DMEM (Gibco) supplement with 10% FBS (Gibco) and 50 µg/mL Primocin (InvivoGen) in 37 °C, 5% CO₂ incubator. For administration of the AhR ligands, 3-MC (Sigma), β-NF (Sigma), Ind (InvivoGen), or Kyn (InvivoGen) were added to the culture media. 3-MC, Ind, and Kyn were dissolved in dimethyl sulfoxide (DMSO, Thermo Fisher Scientific); β-NF was dissolved in acetonitrile (EMD Millipore). The control cells were treated with DMSO.

Preparation of expression vectors and reporter genes

Supplementary Table 1 shows the list of primers used for reporter gene constructs. Each mutant XRE primer includes an EcoRI site to insert the point-mutated XRE. The mutant constructs were generated by modifying p-226 Math1-Luc GL3²⁸ as follows: p-226 II-XRE mut Math1 Luc-GL3: using Math1-5′2 and 3′-111XRE mut as the primer set, the PCR product was obtained from p-226 Math1-Luc GL3 and digested by KpnI and EcoRI. The other PCR product was generated from the same template by using 5′-111XRE mut and Math1-3′2 as the primer set and digested by NcoI and EcoRI. The both fragments were ligated between KpnI and NcoI sites of pGL3 basic (Promega). p-226 I-XRE mut Math1-Luc-GL3: using Math1-5′ -6 XRE mut and Math1-3′ 2 as the primer set, the PCR product was prepared from p-226 Math1-Luc GL3, and digested by NcoI and EcoRI. The other PCR fragment was generated from the same template by using Math1-5′2 and 3′-6XRE mut primers and digested by KpnI and EcoRI. The both fragments were ligated between KpnI and NcoI sites of pGL3 basic. p-226 I, II-XRE mut Math1-Luc-GL3: p-226 II-XRE mut Math1-Luc-GL3 was digested by KpnI and HindIII, and 209 bp of II-XRE mutated fragment was isolated to be cloned into KpnI and HindIII site of p-226 I-XRE mut Math1-Luc-GL3. p-226 I,II-ARE mut Math1-Luc was described previously.²⁸ All constructs were confirmed by sequencing analyses.

Transfection for reporter gene assay

For normalizing transfection efficiency, pRLTKΔARE¹¹ was co-transfected into Hepa 1–6 cells with the battery of Math1 reporter genes using Lipofectamine 2000 (Thermo Fisher Scientific) and Opti-MEM (Gibco) according to the manufacturer’s instructions. Following transfection, the cells were incubated with the indicated AhR ligands, 3-MC (50 nM), β-NF (200 nM), Ind (250 nM), and Kyn (480 nM) or vehicle for 24 h until harvesting. Luciferase activity was measured using a Dual Luciferase assay kit (Promega).

Preparation of expression vectors and nuclear extracts for electrophoresis mobility shift (EMSA) assay

Hind III fragment of mouse AhR cDNA was isolated from pSV SPORT AhR (ATCC, #63125) and cloned into Hind III sites of pcDNA3 (Invitrogen) to prepare the pCMV-mAhR expression vector. pEF Arnt vector was kindly provided from Dr Yasumoto (Tohoku University, Japan). The functional confirmation of the transfected vectors was performed by p4xXRE-tk-Luc reporter gene assay using Hepa 1–6 cells treated with vehicle, 3-MC (2 µM), or β-NF (10 µM). The concentrations of the AhR ligands used in this specific assay were mirrored from previous publications.⁵⁸ The nuclear extracts were prepared from pCMV AhR and pEF Arnt co-transfected HEK293 cells according to a previous publication.⁵⁹ The biotinylated probes were annealed with the complementary oligonucleotide, then subjected to electrophoresis and purified as the double strand probes. For the standard electrophoretic mobility shift assay (EMSA), the reaction mixture containing the probe nucleotides and the nuclear extracts were incubated in buffer (10 mM HEPES (pH 7.9), 25 mM KCl, 1 mM EDTA, 1 mM ZnCl₂, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 4 μg poly(dI-dC), 0.05% Nonidet P-40, and 10% glycerol) at 4 °C for 30 min. For the competition experiments, each competitor oligonucleotide shown in Supplementary Table 2 was added to the standard EMSA reaction mixture at 100-fold molar excess to the probe. For the EMSA supershift assay, the nuclear extracts prepared from Hepa 1–6 cells treated with 3-MC (100 nM) were used and coupled with anti-AhR antibody (NB-100-128, Novus Biologicals). The specificity and selectivity of the antibody was confirmed by Western blot analysis. Detection of EMSA products was performed using the Light Shift Chemiluminescent EMSA Kit (Pierce Thermo Scientific) according to the manufacturer’s instructions. All EMSA experiments were repeated three times utilizing separate nuclear extracts from HEK293 cells.

Chromatin immunoprecipitation (ChIP) assay

Hepa 1–6 cells were treated with vehicle, 3-MC (100 nM), or β-NF (200 nM) for 24 h before formaldehyde cross-linking. ChIP was performed using agarose ChIP Kit (Pierce Thermo Scientific) according to the manufacture’s instructions. Diluted chromatin solution with 2 × 10⁶ cells was incubated with an anti-AhR antibody (NB-100-128, Novus Biologicals) or purified immunoglobulin G (normal goat IgG) (sc-2028, Santa Cruz Biotechnology) for 18 h at 4 °C with rotation. After the DNA was washed and eluted, precipitated DNA was resuspended with 60 µL of water, and 2 µL of DNA was used for PCR amplification with the following primers for Math1 XRE, 5′-Math1-Chip and 3'-Math1-Chip (product size 210 bp), and for Cyp1a1 XRE, 5'-Cyp1a1 Chip and 3'-Cyp1a1 Chip (produced 122 bp)⁶⁰ or 5'1-Ex-Chip Cyp1a1 and 3'-Cyp1a1 Chip (product size 261 bp). All primer sequences are shown in Supplementary Table 3. In the case of the dispersed crypt cells from the mouse intestines, a diluted chromatin solution with 1 × 10⁶ cells were utilized and immunoprecipitated with half scale volume. PCR was conducted with the following condition. For Math1: hot start at 95 °C for 30 s, 94 °C for 30 s, 70 °C for 20 s, 72 °C for 15 s, 28-cylcles for Hepa 1–6 cells or 35-cycles for crypt cell precipitants, respectively. For Cyp1a1: hot start at 95 °C for 30 s, 94 °C for 30 s, 68 °C for 30 s, 72 °C for 20 s, 32-cylcles for Hepa 1–6 cells or 38-cycles for crypt cell precipitants. Image J (National Institutes of Health) was used for quantification analyses.

Immunoblot analysis for MATH1

Hepa 1–6 cells were treated with vehicle, 3-MC (100 nM), or β-NF (200 nM) for 20 h. Lysates from whole nuclei were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. Specific protein signals were detected by anti-ATOH1 antibody (PA5-69557, Invitrogen) and anti-Lamin B1 antibody (#12586, Cell Signaling Technology). Image J (National Institutes of Health) was used for quantification analyses.

Mice

The Vil-Cre (No. 004586), AhR-Flox (No. 006203), and Math1-GFP (No. 013593) mouse lines were obtained from the Jackson Laboratory. All mice used in the experiments were in the albino C57BL/6J background (B6 (Cg)-Tyrc-2J/J), male, 8–10 weeks old. AhR^F/F::VilCre mice were generated by crossing AhR^F/F::VilCre and AhR^F/F. Homozygous Math1-GFP mice were used for mating and for experiments. All mice were housed in a pathogen-free animal facility and handled according to the guidelines of the National Institutes of Health. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Fred Hutchinson Cancer Center.

β-NF treatment for in vivo ChIP assay

Wild-type mice were treated with a single dose of β-NF (Sigma) dissolved in 8% DMSO, 92% corn oil (Thermo Fisher Scientific) (40 mg/kg BW, i.p.), or vehicle alone. Small intestines were harvested after 5 h of treatment. Intestinal crypts were isolated as described previously.⁶¹ Single-cell suspensions were prepared using TrypLE Express (Gibco) for ChIP assay.

Organoid culture

Mouse intestinal organoids were established and maintained as described previously,⁶² with modification. Briefly, a segment of the proximal intestine (approximately 23 cm) was harvested, washed with cold PBS, and opened up longitudinally to expose the luminal surface. Intestinal crypts were isolated using the dissociation regent (STEMCELL), then cultured in a mixture of Matrigel (BD Biosciences) and the culture medium designed for mouse intestinal organoids (STEMCELL) under standard conditions (5% CO², 37 °C). Based on our preliminary experiment, crypt isolation from AhR^F/F::VilCre mouse was performed using 5 mM EDTA/PBS instead of the dissociation regent (STEMCELL) used for wild-type mice. The treatment of organoids was performed with media supplemented with 10 µM of DAPT (Calbiochem, dissolved in DMSO) and/or 5 nM or 50 nM of β-NF (Sigma, dissolved in acetonitrile) for the indicated time period with media change every day. The control treatment was performed using the corresponding vehicle for DAPT or β-NF. All experiments in this study were performed using primary intestinal organoids without any passages. After culture, the organoids were harvested for RNA extraction or histological analysis.

Sequential treatment of Math1-GFP report mice

Homogenous Math1-GFP mice were treated with a single dose of DBZ (30 µmol/kg BW, i.p., LY-41157, Sigma) dissolved in 10% DMSO, 90% vehicle solution (0.5% hydroxypropylmethyl cellulose (Sigma) and 0.1% Tween 80 (Thermo Fisher Scientific) in water), followed by three doses of β-NF (40 mg/kg BW, i.p., Sigma) dissolved in 8% DMSO, 92% corn oil (Thermo Fisher Scientific), or vehicle alone. Small intestines were harvested for histological analysis 24 h after the last treatment.

RNA preparation and quantitative real time-PCR (qPCR)

The organoids and intestinal mucosal tissues were homogenized in TRIzol (Thermo Fisher Scientific), and total RNA was extracted as previously reported.⁶³ cDNA was synthesized using the qScript system (Quanta Biosciences) and a multiplex PCR preamplification of specific cDNA targets and endogenous control was performed using TaqMan PreAmp Master Mix (Applied Biosystems) following the manufacturer’s instructions. qPCR was performed by QuantStudio 7 (Applied Biosystems) with TaqMan Fast Advanced Master Mix (Applied Biosystems). The primers are shown in Supplementary Table 4. Expression levels of each gene were normalized to Actb and calculated relative to the control.

Histology and immunofluorescence

Intestinal organoids were fixed in 4% paraformaldehyde at room temperature for 1 h followed by agarose gel and paraffin embedding as previously reported⁶⁴ to get 4 µm thick paraffin sections. Small intestines were placed in 4% paraformaldehyde at 4 °C overnight and then cut length wise, rolled, and paraffin embedded. For detection of goblet cells, the Alcian blue staining kit (ab150662, abcam) was used. For immunohistochemistry, goat anti-GFP (1:200, ab6673, abcam), rat anti-Ki67 (1:100, 652401, BioLegend), rabbit anti-Chromogranin A (1:500, ab15160, abcam), rabbit anti-Lysozyme (1:1000, ab108508, Abcam), and rabbit anti-MUC2 (1:50, PA579702, Invitrogen) were used as the primary antibodies. The following secondary antibodies were used, respectively: Cy3 donkey anti-goat (1:200, Jackson ImmunoResearch), Alexa Fluor 488 donkey anti-rat (1:200, Thermo Fisher Scientific), Alexa Fluor 488 anti-rabbit 488 (1:200, Thermo Fisher Scientific). Imaging data were collected using a microscope (Eclipse E800, Nikon) and a camera (AxioCam Mrc, Zeiss) for blight field images of the organoids. Fluorescent image data of the intestinal sections were collected using a microscope (Imager Z2, Zeiss) and a fluorescence scanning system (TissueFAXS, TissueGnostics). A confocal microscope system (Andor Dragonfly Spinning Disk, Oxford Instruments) and a camera (iXon EMCCD, Oxford Instruments) were used for fluorescent image data of the intestinal organoids, and z-stacks were acquired at optimal stack distance. The quantification of goblet cells was performed by using ImageJ (National Institutes of Health), where Alcian blue-stained positive cells in each organoid were counted by examining at least 60 organoids from one section and normalized by the cross-sectional area of each organoid. The quantification of Math1-GFP and Ki67-positive cells in the organoids and crypts in the small intestine was performed using Imaris (Oxford Instruments). The number of GFP positive cells, Ki67 positive cells, and GFP and Ki67 double-positive cells in the organoids was counted by examining at least 25 organoids from each experiment and normalized by the number of DAPI stained cells. The quantification of crypt epithelium was performed by examining approximately 20% of the anterior region of the small intestine, where at least 50 of well-oriented crypts were analyzed per section.

Statistical analysis

GraphPad Prism 9 software was used for statistical analysis of data sets. Quantitative data are presented as mean ± SD. For the comparison of two groups, unpaired two-tailed Student’s t test was used; for more than two groups, one- or two-way ANOVA was used followed by Tukey's test, as described in the relevant figure legends.

Funding Statement

This work was supported by the National Institutes of Health [R35 CA197222, to TWK] and the Washington State Andy Hill CARE Fund [to TWK]. This research was also supported by the Experimental Histopathology and Cellular Imaging Core of the Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium [P30 CA015704].

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2022.2160610.

AUTHOR CONTRIBUTIONS

YY designed and performed all intestinal organoid related experiments, analyzed data, and drafted the manuscript. TJ contributed to mouse genotyping, organoid preparation and Alcian blue staining. TWK directed and coordinated the study and contributed to manuscript preparation. NW performed all gene regulation experiments and co-directed the research, and contributed to manuscript preparation. All authors reviewed the final version.