Murine intestinal stem cells are highly sensitive to modulation of the T3/TRα1-dependent pathway.

The thyroid hormone T3 and its nuclear receptor TRα1 control gut development and homeostasis through the modulation of intestinal crypt cell proliferation. Despite increasing data, in-depth analysis on their specific action on intestinal stem cells is lacking. By using ex vivo 3D organoid cultures and molecular approaches, we observed early responses to T3 involving the T3-metabolizing enzyme Dio1 and the transporter Mct10, accompanied by a complex response of stem cell- and progenitor-enriched genes. Interestingly, specific TRα1 loss-of-function (inducible or constitutive) was responsible for low ex vivo organoid development and impaired stem cell activity. T3 treatment of animals in vivo not only confirmed the positive action of this hormone on crypt cell proliferation but also demonstrated its key action in modulating the number of stem cells, the expression of their specific markers and the commitment of progenitors into lineage-specific differentiation. In conclusion, T3 treatment or TRα1 modulation has a rapid and strong effect on intestinal stem cells, broadening our perspectives in the study of T3/TRα1-dependent signaling in these cells.

Thyroid hormone signaling in the intestinal stem cells and their niche.

Several studies emphasized the function of the thyroid hormones in stem cell biology. These hormones act through the nuclear hormone receptor TRs, which are T3-modulated transcription factors. Pioneer work on T3-dependent amphibian metamorphosis showed that the crosstalk between the epithelium and the underlying mesenchyme is absolutely required for intestinal maturation and stem cell emergence. With the recent advances of powerful animal models and 3D-organoid cultures, similar findings have now begun to be described in mammals, where the action of T3 and TRα1 control physiological and cancer-related stem cell biology. In this review, we have summarized recent findings on the multiple functions of T3 and TRα1 in intestinal epithelium stem cells, cancer stem cells and their niche. In particular, we have highlighted the regulation of metabolic functions directly linked to normal and/or cancer stem cell biology. These findings help explain other possible mechanisms by which TRα1 controls stem cell biology, beyond the more classical Wnt and Notch signaling pathways.

MAVS deficiency induces gut dysbiotic microbiota conferring a proallergic phenotype.

Prominent changes in the gut microbiota (referred to as "dysbiosis") play a key role in the development of allergic disorders, but the underlying mechanisms remain unknown. Study of the delayed-type hypersensitivity (DTH) response in mice contributed to our knowledge of the pathophysiology of human allergic contact dermatitis. Here we report a negative regulatory role of the RIG-I-like receptor adaptor mitochondrial antiviral signaling (MAVS) on DTH by modulating gut bacterial ecology. Cohousing and fecal transplantation experiments revealed that the dysbiotic microbiota of Mavs-/- mice conferred a proallergic phenotype that is communicable to wild-type mice. DTH sensitization coincided with increased intestinal permeability and bacterial translocation within lymphoid organs that enhanced DTH severity. Collectively, we unveiled an unexpected impact of RIG-I-like signaling on the gut microbiota with consequences on allergic skin disease outcome. Primarily, these data indicate that manipulating the gut microbiota may help in the development of therapeutic strategies for the treatment of human allergic skin pathologies.

Deletion of Stearoyl-CoA Desaturase-1 From the Intestinal Epithelium Promotes Inflammation and Tumorigenesis, Reversed by Dietary Oleate.

BACKGROUND & AIMS: The enzyme stearoyl-coenzyme A desaturase 1 (SCD or SCD1) produces monounsaturated fatty acids by introducing double bonds into saturated bonds between carbons 9 and 10, with oleic acid as the main product. SCD1 is present in the intestinal epithelium, and fatty acids regulate cell proliferation, so we investigated the effects of SCD1-induced production of oleic acid in enterocytes in mice. METHODS: We generated mice with disruption of Scd1 selectively in the intestinal epithelium (iScd1-/- mice) on a C57BL/6 background; iScd1+/+ mice were used as controls. We also generated iScd1-/-ApcMin/+ mice and studied cancer susceptibility. Mice were fed a chow, oleic acid-deficient, or oleic acid-rich diet. Intestinal tissues were collected and analyzed by histology, reverse transcription quantitative polymerase chain reaction, immunohistochemistry, and mass spectrometry, and tumors were quantified and measured. RESULTS: Compared with control mice, the ileal mucosa of iScd1-/- mice had a lower proportion of palmitoleic (C16:1 n-7) and oleic acids (C18:1 n-9), with accumulation of stearic acid (C18:0); this resulted a reduction of the Δ9 desaturation ratio between monounsaturated (C16:1 n-7 and C18:1 n-9) and saturated (C16:0 and C18:0) fatty acids. Ileal tissues from iScd1-/- mice had increased expression of markers of inflammation activation and crypt proliferative genes compared with control mice. The iScd1-/-ApcMin/+ mice developed more and larger tumors than iScd1+/+ApcMin/+ mice. iScd1-/-ApcMin/+ mice fed the oleic acid-rich diet had reduced intestinal inflammation and significantly lower tumor burden compared with mice fed a chow diet. CONCLUSIONS: In studies of mice, we found intestinal SCD1 to be required for synthesis of oleate in the enterocytes and maintenance of fatty acid homeostasis. Dietary supplementation with oleic acid reduces intestinal inflammation and tumor development in mice.

The thyroid hormone nuclear receptors and the Wnt/ß-catenin pathway: An intriguing liaison.

The thyroid hormones, T3 and T4, control several developmental and homeostatic processes. From a molecular point of view, most of their actions depend on the activity of the thyroid hormone nuclear receptors (TRs), which are T3-modulated transcription factors. Recent studies have not only highlighted that the physiological response induced by T3 within a cell depends on the expression of specific TRs, but also that the functions of TRs are coordinated by and integrated in other signalling pathways. This is particularly the case for the multilevel interactions between TRs and the Wnt signalling pathway. Interestingly both signals are involved in development and homeostasis, and their alterations are responsible for the development of pathologies, such as cancer. Here, we present findings on the complex crosstalk between TRs and Wnt in several organisms and in different tissue contexts, and speculate on the biological relevance of modulating TR-Wnt functionality in therapeutic approaches aimed to target cancer cells or applications for regenerative medicine.

The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

WNT/ß-catenin signalling is activated in aldosterone-producing adenomas and controls aldosterone production.

Primary aldosteronism (PA) is the main cause of secondary hypertension, resulting from adrenal aldosterone-producing adenomas (APA) or bilateral hyperplasia. Here, we show that constitutive activation of WNT/ß-catenin signalling is the most frequent molecular alteration found in 70% of APA. We provide evidence that decreased expression of the WNT inhibitor SFRP2 may be contributing to deregulated WNT signalling and APA development in patients. This is supported by the demonstration that mice with genetic ablation of Sfrp2 have increased aldosterone production and ectopic differentiation of zona glomerulosa cells. We further show that ß-catenin plays an essential role in the control of basal and Angiotensin II-induced aldosterone secretion, by activating AT1R, CYP21 and CYP11B2 transcription. This relies on both LEF/TCF-dependent activation of AT1R and CYP21 regulatory regions and indirect activation of CYP21 and CYP11B2 promoters, through increased expression of the nuclear receptors NURR1 and NUR77. Altogether, these data show that aberrant WNT/ß-catenin activation is associated with APA development and suggest that WNT pathway may be a good therapeutic target in PA.

The secreted Frizzled-Related Protein 2 modulates cell fate and the Wnt pathway in the murine intestinal epithelium.

The secreted Frizzled-Related Proteins (sFRPs) are generally considered antagonistic to Wnt signaling. However, several studies have described their synergy and/or activation of this pathway. Our own data indicated that in the intestinal epithelium, thyroid hormone induced-expression of sFRP2 stabilizes ß-catenin, leading to induction of Wnt. The aim of this work was to investigate the role of sFRP2 in the intestinal epithelium homeostasis and its specific effect on canonical Wnt pathway. In wild type animals we observed a restricted pattern of sFRP2 protein expression at the level of the intestinal crypts. Interestingly, sFRP2(-/-) mice displayed increased apoptosis within the crypts together with a defect in cell migration. Because of altered proportion of lineage-specific committed progenitors, the sFRP2(-/-) animals also showed a decrease of absorptive differentiation counterbalanced by an increase of secretory differentiation. Regarding the action of sFRP2 on canonical Wnt pathway, the lack of sFRP2 expression in sFRP2(-/-)/TopGal animals in vivo reduced the Wnt activity. This positive action of sFRP2 on Wnt was further confirmed by in vitro studies. In conclusion, in accordance with its restricted expression profile, sFRP2 contributes to the physiology of the intestinal epithelial crypt progenitors by controlling apoptosis, cell fate decisions and the Wnt pathway.

Telomere protection and TRF2 expression are enhanced by the canonical Wnt signalling pathway.

The DNA-binding protein TRF2 is essential for telomere protection and chromosome stability in mammals. We show here that TRF2 expression is activated by the Wnt/ß-catenin signalling pathway in human cancer and normal cells as well as in mouse intestinal tissues. Furthermore, ß-catenin binds to TRF2 gene regulatory regions that are functional in a luciferase transactivating assay. Reduced ß-catenin expression in cancer cells triggers a marked increase in telomere dysfunction, which can be reversed by TRF2 overexpression. We conclude that the Wnt/ß-catenin signalling pathway maintains a level of TRF2 critical for telomere protection. This is expected to have an important role during development, adult stem cell function and oncogenesis.

Thyroid hormones and their nuclear receptors: new players in intestinal epithelium stem cell biology?

Thyroid hormones participate in the development and homeostasis of several organs and tissues. It is well documented that they act via nuclear receptors, the TRs, which are transcription factors whose function is modulated by the hormone T3. Importantly, T3-induced physiological response within a cell depends on the specific TR expression and on the T3 bioavailability. However, in addition to this T3-dependent control of TR functionality, increasing data show that the action of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. By focusing on the intestinal epithelium of both amphibians and mammals we summarize here new data in support of a role for thyroid hormones and the TR nuclear receptors in stem cell biology. This new concept may be extended to other organs and have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer.

CDX2 regulation by the RNA-binding protein MEX3A: impact on intestinal differentiation and stemness.

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis.

Thyroid hormone's action on progenitor/stem cell biology: new challenge for a classic hormone?

BACKGROUND: Thyroid hormones are involved in developmental and homeostatic processes in several tissues. Their action results in different outcomes depending on the developmental stage, tissue and/or cellular context. Interestingly, their pleiotropic roles are conserved across vertebrates. It is largely documented that thyroid hormones act via nuclear receptors, the TRs, which are transcription factors and whose activity can be modulated by the local availability of the hormone T3. In the "classical view", the T3-induced physiological response depends on the expression of specific TR isoforms and the iodothyronine deiodinase selenoenzymes that control the local level of T3, thus TR activity. SCOPE OF THE REVIEW: Recent data have clearly established that the functionality of TRs is coordinated and integrated with other signaling pathways, specifically at the level of stem/progenitor cell populations. Here, we summarize these data and propose a new and intriguing role for thyroid hormones in two selected examples. MAJOR CONCLUSIONS: In the intestinal epithelium and the retina, TRα1 and TRß2 are expressed at the level of the precursors where they induce cell proliferation and differentiation, respectively. Moreover, these different functions result from the integration of the hormone signal with other intrinsic pathways, which play a fundamental role in progenitor/stem cell physiology. GENERAL SIGNIFICANCE: Taken together, the interaction of TRs with other signaling pathways, specifically in stem/progenitor cells, is a new concept that may have biological relevance in therapeutic approaches aimed to target stem cells such as tissue engineering and cancer. This article is part of a Special Issue entitled Thyroid hormone signalling.

Brief report: musashi1-eGFP mice, a new tool for differential isolation of the intestinal stem cell populations.

The intestinal epithelium self-renews rapidly and continuously throughout life, due to the presence of crypt stem cells. Two pools of these cells have been identified in the small intestine, which differ in position ("+4" or the bottom of the crypts), expression of specific markers (Bmi1/mTert or Lgr5/Ascl2), and cell cycle characteristics. Interestingly, the RNA-binding protein Musashi1 is expressed in both populations and therefore a potential marker for both stem cell types. In order to locate, isolate, and study Musashi1-expressing cells within the intestinal epithelium, we generated transgenic mice expressing GFP fluorescent protein under the control of a 7-kb Msi1 promoter. The expression pattern of GFP in the intestinal crypts of both small and large intestines completely overlapped that of Musashi1, validating our model. By using fluorescence-activated cell sorting, cellular, and molecular analyses, we showed that GFP-positive Msi1-expressing cells are divided into two major pools corresponding to the Lgr5- and mTert-expressing stem cells. Interestingly, monitoring the cell cycle activity of the two sorted populations reveals that they are both actively cycling, although differences in cell cycle length were confirmed. Altogether, our new reporter mouse model based upon Musashi1 expression is a useful tool to isolate and study stem cells of the intestinal epithelium. Moreover, these mice uniquely enable the concomitant study of two pools of intestinal stem cells within the same animal model.

Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

General transcription factor TAF4 antagonizes epigenetic silencing by Polycomb to maintain intestine stem cell functions.

Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.

The thyroid hormones and their nuclear receptors in the gut: from developmental biology to cancer.

The thyroid hormones control the development and the homeostasis of several organs in vertebrates. Their actions depend, for the most part, on nuclear receptors, the TRs, which are transcription factors whose activity is modulated by the hormone T3. The gastrointestinal tract is a well characterized target of thyroid hormones and TRs, as extensively described in the literature. In fact, its remodeling in amphibians during thyroid hormone-dependent metamorphosis is well characterized at the cellular and the molecular levels. However, whereas a great attention has been paid to the nervous system and to cardiac development and physiology, the function of thyroid hormones and TRs in the mammalian gastrointestinal tract has been, until recently, underestimated. Several studies have described an important conservation of this hormonal signal during intestinal development and have suggested that it may play a role in stem cell physiology in both amphibians and mammals. These findings show the importance of the thyroid hormones and TRs, whose homologous actions are maintained across species. In the present review, we summarize the most recent data on this issue, starting from work that has been conducted on amphibian metamorphosis to results on postnatal development, homeostasis, and tumorigenesis in mammals. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

The overexpression of the putative gut stem cell marker Musashi-1 induces tumorigenesis through Wnt and Notch activation.

The RNA-binding protein Musashi-1 (Msi1) has been proposed as a marker of intestinal epithelial stem cells. These cells are responsible for the continuous renewal of the intestinal epithelium. Although the function of Msi1 has been studied in several organs from different species and in mammalian cell lines, its function and molecular regulation in mouse intestinal epithelium progenitor cells are still undefined. We describe here that, in these cells, the expression of Msi1 is regulated by the canonical Wnt pathway, through a mechanism involving a functional Tcf/Lef binding site on its promoter. An in vitro study in intestinal epithelium primary cultures showed that Msi1 overexpression promotes progenitor proliferation and activates Wnt and Notch pathways. Moreover, Msi1-overexpressing cells exhibit tumorigenic properties in xenograft experiments. These data point to a positive feedback loop between Msi1 and Wnt in intestinal epithelial progenitors. They also suggest that Msi1 has oncogenic properties in these cells, probably through induction of both the Wnt and Notch pathways.

Thyroid Hormone Signaling and Function: News from Classical and Emerging Models.

According to Brown and Cai, Thyroid hormones (THs) have been considered "the first developmental morphogen ever discovered" [...].

Regulation of the THRA gene, encoding the thyroid hormone nuclear receptor TRα1, in intestinal lesions.

The THRA gene, encoding the thyroid hormone nuclear receptor TRα1, is expressed in an increasing gradient at the bottom of intestinal crypts, overlapping with high Wnt and Notch activities. Importantly, THRA is upregulated in colorectal cancers, particularly in the high-Wnt molecular subtype. The basis of this specific and/or altered expression pattern has remained unknown. To define the mechanisms controlling THRA transcription and TRα1 expression, we used multiple in vitro and ex vivo approaches. Promoter analysis demonstrated that transcription factors important for crypt homeostasis and altered in colorectal cancers, such as transcription factor 7-like 2 (TCF7L2; Wnt pathway), recombining binding protein suppressor of hairless (RBPJ; Notch pathway), and homeobox protein CDX2 (epithelial cell identity), modulate THRA activity. Specifically, although TCF7L2 and CDX2 stimulated THRA, RBPJ induced its repression. In-depth analysis of the Wnt-dependent increase showed direct regulation of the THRA promoter in cells and of TRα1 expression in murine enteroids. Given our previous results on the control of the Wnt pathway by TRα1, our new results unveil a complex regulatory loop and synergy between these endocrine and epithelial-cell-intrinsic signals. Our work describes, for the first time, the regulation of the THRA gene in specific cell and tumor contexts.

Cooperation between the thyroid hormone receptor TRalpha1 and the WNT pathway in the induction of intestinal tumorigenesis.

BACKGROUND & AIMS: Colorectal tumorigenesis is a multistep process involving the alteration of oncogenes and tumor suppressor genes, leading to the deregulation of molecular pathways that govern intestinal homeostasis. We have previously shown that the thyroid hormone receptor alpha1 (TRalpha1) controls intestinal development and homeostasis through the WNT pathway. More precisely, TRalpha1 directly enhances the transcription of several components of this pathway, allowing increased expression of beta-catenin/Tcf4 target genes and stimulation of cell proliferation. Because the WNT pathway is a major player in controlling intestinal homeostasis, we addressed whether the TRalpha1 receptor has tumor-inducing potential. METHODS: We generated mice overexpressing TRalpha1 specifically in the intestinal epithelium in a wild-type (vil-TRalpha1) or a WNT-activated (vil-TRalpha1/Apc(+/1638N)) genetic background. RESULTS: The intestine of vil-TRalpha1 mice presents aberrant intestinal mucosal architecture and increased cell proliferation and develops adenoma at a low rate. However, TRalpha1 overexpression is unable to induce cancer development. On the contrary, we observed accelerated tumorigenesis in vil-TRalpha1/Apc(+/1638N) mice compared with the Apc(+/1638N) mutants. CONCLUSION: Our results suggest that this phenotype is due to cooperation between the activated TRalpha1 and WNT pathways. This is the first report describing the tumor-inducing function of TRalpha1 in the intestine.