Optogenetically controlled inflammasome activation demonstrates two phases of cell swelling during pyroptosis.

Inflammasomes are multiprotein platforms that control caspase-1 activation, which process the inactive precursor forms of the inflammatory cytokines IL-1ß and IL-18, leading to an inflammatory type of programmed cell death called pyroptosis. Studying inflammasome-driven processes, such as pyroptosis-induced cell swelling, under controlled conditions remains challenging because the signals that activate pyroptosis also stimulate other signaling pathways. We designed an optogenetic approach using a photo-oligomerizable inflammasome core adapter protein, apoptosis-associated speck-like containing a caspase recruitment domain (ASC), to temporally and quantitatively manipulate inflammasome activation. We demonstrated that inducing the light-sensitive oligomerization of ASC was sufficient to recapitulate the classical features of inflammasomes within minutes. This system showed that there were two phases of cell swelling during pyroptosis. This approach offers avenues for biophysical investigations into the intricate nature of cellular volume control and plasma membrane rupture during cell death.

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.

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.

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.

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.

Analysis of virion-incorporated host proteins required for herpes simplex virus type 1 infection through a RNA interference screen.

Viruses are strictly dependent on cells to propagate and many incorporate host proteins in their viral particles, but the significance of this incorporation is poorly understood. Recently, we performed the first comprehensive characterization of the mature herpes simplex virus type 1 (HSV-1) in which up to 49 distinct cellular proteins were identified by mass spectrometry. In the present study, we sought to identify if these cellular factors are relevant for the HSV-1 life cycle. To this end, we performed a small interfering RNA functional screen and found that 15 of these host proteins altered HSV-1 proliferation in cell culture, without any significant effect on cell viability. Moreover, the siRNA used had no negative consequences for Adenovirus type 5 propagation (with one exception) indicating that the modulation was specific for HSV-1 and not merely due to unhealthy cells. The positive host proteins include several Rab GTPases and other intracellular transport components as well as proteins involved in signal transduction, gene regulation and immunity. Remarkably, in most cases when virions were depleted for one of the above proteins, they replicated more poorly in subsequent infections in wild type cells. This highlights for the first time that both the cellular and virion-associated pools of many of these proteins actively contribute to viral propagation. Altogether, these findings underscore the power and biological relevance of combining proteomics and RNA interference to identify novel host-pathogen interactions.

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.

Multi-level interactions between the nuclear receptor TRα1 and the WNT effectors ß-catenin/Tcf4 in the intestinal epithelium.

Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of ß-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of ß-catenin/Tcf4. Moreover, TRα1 physically interacted with ß-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased ß-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.

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.

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.

The frizzled-related sFRP2 gene is a target of thyroid hormone receptor alpha1 and activates beta-catenin signaling in mouse intestine.

The thyroid hormone receptor TRalpha1 regulates intestinal development and homeostasis by controlling epithelial proliferation in the crypts. This involves positive control of the Wnt/beta-catenin pathway. To further investigate the effect of thyroid hormone-TRalpha1 signaling on the intestinal epithelium proliferating compartment, we performed a comparative transcription profile analysis on laser microdissected crypt cells recovered from wild type animals with normal or perturbed hormonal status, as well as from TR knock-out mice. Statistical analysis and an in silico approach allowed us to identify 179 differentially regulated genes and to group them into organized functional networks. We focused on the "cell cycle/cell proliferation" network and, in particular, on the Frizzled-related protein sFRP2, whose expression was greatly increased in response to thyroid hormones. In vitro and in vivo analyses showed that the expression of sFRP2 is directly regulated by TRalpha1 and that it activates beta-catenin signaling via Frizzled receptors. Indeed, sFRP2 stabilizes beta-catenin, activates its target genes, and enhances cell proliferation. In conclusion, these new data, in conjunction with our previous results, indicate a complex interplay between TRalpha1 and components of the Wnt/beta-catenin pathway. Moreover, we describe in this study a novel mechanism of action of sFRP2, responsible for the activation of beta-catenin signaling.

The thyroid hormone receptor-alpha (TRalpha) gene encoding TRalpha1 controls deoxyribonucleic acid damage-induced tissue repair.

The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-alpha1 (TRalpha1), intestinal crypt cell proliferation in the mouse. In order to understand whether this receptor also plays a role in intestinal regeneration after DNA damage, we applied a protocol of gamma-ray irradiation and monitored cell proliferation and apoptosis at several time points. In wild-type mice, the dose of 8 Gy induced cell cycle arrest and apoptosis in intestinal crypts a few hours after irradiation. This phenomenon reverted 48 h after irradiation. TRalpha(0/0) mutant mice displayed a constant low level of proliferating cells and a high apoptosis rate during the period of study. At the molecular level, in TRalpha(0/0) animals we observed a delay in the p53 phosphorylation induced by DNA damage. In our search for the expression of the protein kinases responsible for p53 phosphorylation upon irradiation, we have focused on DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The number of cells expressing DNA-PKcs in crypts remained high 48 h after irradiation, specifically in TRalpha mutants. Altogether, in TRalpha(0/0) animals the rate of apoptosis in crypt cells remained high, apparently due to an elevated number of cells still presenting DNA damage. In conclusion, the TRalpha gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.