Cellular and molecular gastroenterology and hepatology
Li, C;Zhou, Y;Wei, R;Napier, DL;Sengoku, T;Alstott, MC;Liu, J;Wang, C;Zaytseva, YY;Weiss, HL;Wang, Q;Evers, BM;
PMID: 36584817 | DOI: 10.1016/j.jcmgh.2022.12.012
The Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined.Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 MAPK, the transcription factor atonal homolog 1 (ATOH1), and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by western blot, qPCR or IF and IHC staining.HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout (KO) or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 KO resulted in activation of p38 MAPK and increased expression of ATOH1; inhibition of p38 MAPK signaling attenuated the phenotypes induced by HK2 KO in intestinal organoids. HK2 KO significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 KO.Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 MAPK/ATOH1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.
Frontiers in molecular neuroscience
Kim, JJ;Sapio, MR;Vazquez, FA;Maric, D;Loydpierson, AJ;Ma, W;Zarate, CA;Iadarola, MJ;Mannes, AJ;
PMID: 35706427 | DOI: 10.3389/fnmol.2022.892345
Ketamine, an N-methyl-D-aspartate (NMDA)-receptor antagonist, is a recently revitalized treatment for pain and depression, yet its actions at the molecular level remain incompletely defined. In this molecular-pharmacological investigation in the rat, we used short- and longer-term infusions of high dose ketamine to stimulate neuronal transcription processes. We hypothesized that a progressively stronger modulation of neuronal gene networks would occur over time in cortical and limbic pathways. A continuous intravenous administration paradigm for ketamine was developed in rat consisting of short (1 h) and long duration (10 h, and 10 h + 24 h recovery) infusions of anesthetic concentrations to activate or inhibit gene transcription in a pharmacokinetically controlled fashion. Transcription was measured by RNA-Seq in three brain regions: frontal cortex, hippocampus, and amygdala. Cellular level gene localization was performed with multiplex fluorescent in situ hybridization. Induction of a shared transcriptional regulatory network occurred within 1 h in all three brain regions consisting of (a) genes involved in stimulus-transcription factor coupling that are induced during altered synaptic activity (immediate early genes, IEGs, such as c-Fos, 9-12 significant genes per brain region, p < 0.01 per gene) and (b) the Nrf2 oxidative stress-antioxidant response pathway downstream from glutamate signaling (Nuclear Factor Erythroid-Derived 2-Like 2) containing 12-25 increasing genes (p < 0.01) per brain region. By 10 h of infusion, the acute results were further reinforced and consisted of more and stronger gene alterations reflecting a sustained and accentuated ketamine modulation of regional excitation and plasticity. At the cellular level, in situ hybridization localized up-regulation of the plasticity-associated gene Bdnf, and the transcription factors Nr4a1 and Fos, in cortical layers III and V. After 24 h recovery, we observed overshoot of transcriptional processes rather than a smooth return to homeostasis suggesting an oscillation of plasticity occurs during the transition to a new phase of neuronal regulation. These data elucidate critical molecular regulatory actions during and downstream of ketamine administration that may contribute to the unique drug actions of this anesthetic agent. These molecular investigations point to pathways linked to therapeutically useful attributes of ketamine.
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
Stanisavljević L, Myklebust MP, Leh S, Dahl O.
PMID: 27435662 | DOI: 10.1080/0284186X.2016.1201215
Abstract
BACKGROUND:
Expression of leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) gene is associated with a metastatic phenotype and poor prognosis in colorectal cancer (CRC). CD133 expression is a putative cancer stem cell marker and a proposed prognostic marker in CRC, whereas the predictive value of CD133 expression for effect of adjuvant chemotherapy in CRC is unclear.
MATERIAL AND METHODS:
For the study of LGR5 mRNA and CD133 expression, tissue microarrays from 409 primary CRC stage II and III tumors, where patients had been randomized to adjuvant chemotherapy or surgery only, were available. LGR5 mRNA and CD133 expression were assessed by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. LGR5 mRNA and CD133 expression as prognostic and predictive markers were evaluated by univariate and multivariate analyses.
RESULTS:
For all CRC patients, positive LGR5 mRNA and CD133 expression were associated with classic adenocarcinoma histology type (p = 0.001 and p = 0.014, respectively). Positive LGR5 mRNA expression was also associated with smaller tumor diameter for CRC stage II (p = 0.005), but not for CRC stage III (p = 0.054). For CRC stage II, lack of LGR5 mRNA expression was associated with longer time to recurrence (TTR) in Kaplan-Meier (p = 0.045) and in multivariate Cox analysis (HR 0.27, 95% CI 0.08-0.95, p = 0.041). For colon cancer stage III patients, lack of CD133 expression was associated with better effect of adjuvant chemotherapy (p = 0.016) in Kaplan-Meier univariate analysis, but the interaction between CD133 and adjuvant chemotherapy was not statistically significant in multivariate analysis (HR 0.59, 95% CI 0.18-1.89, p = 0.374).
CONCLUSION:
LGR5 mRNA expression is a prognostic factor for CRC stage II patients, whereas the value of CD133 expression as prognostic and predictive biomarker is inconclusive.
Cellular and Molecular Gastroenterology and Hepatology
Tsai YH, Hill DR, Kumar N, Huang S, Chin AM, Dye BR, Nagy MS, Verzi MP, Spence JR.
PMID: - | DOI: 10.1016/j.jcmgh.2016.06.002
Background & Aims
The Lgr family of transmembrane proteins (Lgr4, 5, 6) act as functional receptors for R-spondin proteins (Rspo 1, 2, 3, 4), and potentiate Wnt signaling in different contexts. Lgr5 is arguably the best characterized of the Lgr family members in a number of adult and embryonic of contexts in mice. However, the function ofLGR family members in early embryonic development is unclear, and has not been explored during human development or tissue differentiation in detail.
Methods
We interrogated the function and expression of LGR family members using human pluripotent stem cell–derived tissues including definitive endoderm, mid/hindgut, and intestinal organoids. We performed embryonic lineage tracing in Lgr5–creER–eGFP mice.
Results
We show that LGR5 is part of the human definitive endoderm (DE) gene signature, and LGR5 transcripts are induced robustly when human pluripotent stem cells are differentiated into DE. Our results show that LGR4and 5 are functionally required for efficient human endoderm induction. Consistent with data in human DE, we observe Lgr5 reporter (eGFP) activity in the embryonic day 8.5 mouse endoderm, and show the ability to lineage trace these cells into the adult intestine. However, gene expression data also suggest that there are human–mouse species-specific differences at later time points of embryonic development.
Conclusions
Our results show that LGR5 is induced during DE differentiation, LGR receptors are functionally required for DE induction, and that they function to potentiate WNT signaling during this process.
Toxicol Sci. 2018 Oct 26.
Peters MF, Landry T, Pin C, Maratea K, Dick C, Wagoner MP, Choy AL, Barthlow H, Snow D, Stevens Z, Armento A, Scott CW, Ayehunie S.
PMID: 30364994 | DOI: 10.1093/toxsci/kfy268
Drug-induced gastrointestinal toxicities (GITs) rank among the most common clinical side effects. Preclinical efforts to reduce incidence are limited by inadequate predictivity of in vitro assays. Recent breakthroughs in in vitro culture methods support intestinal stem cell maintenance and continual differentiation into the epithelial cell types resident in the intestine. These diverse cells self-assemble into microtissues with in vivo-like architecture. Here, we evaluate human GI microtissues grown in transwell plates that allow apical and/or basolateral drug treatment and 96-well throughput. Evaluation of assay utility focused on predictivity for diarrhea since this adverse effect correlates with intestinal barrier dysfunction which can be measured in GI microtissues using transepithelial electrical resistance (TEER). A validation set of widely prescribed drugs was assembled and tested for effects on TEER. When the resulting TEER inhibition potencies were adjusted for clinical exposure, a threshold was identified that distinguished drugs that induced clinical diarrhea from those that lack this liability. Microtissue TEER assay predictivity was further challenged with a smaller set of drugs whose clinical development was limited by diarrhea that was unexpected based on one-month animal studies. Microtissue TEER accurately predicted diarrhea for each of these drugs. The label-free nature of TEER enabled repeated quantitation with sufficient precision to develop a mathematical model describing the temporal dynamics of barrier damage and recovery. This human 3D GI microtissue is the first in vitro assay with validated predictivity for diarrhea-inducing drugs. It should provide a platform for lead optimization and offers potential for dose schedule exploration.
Garcia-Alonso, L;Lorenzi, V;Mazzeo, CI;Alves-Lopes, JP;Roberts, K;Sancho-Serra, C;Engelbert, J;Marečková, M;Gruhn, WH;Botting, RA;Li, T;Crespo, B;van Dongen, S;Kiselev, VY;Prigmore, E;Herbert, M;Moffett, A;Chédotal, A;Bayraktar, OA;Surani, A;Haniffa, M;Vento-Tormo, R;
PMID: 35794482 | DOI: 10.1038/s41586-022-04918-4
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
Lim, HYG;Yada, S;Barker, N;
PMID: 35620071 | DOI: 10.1016/j.xpro.2022.101411
Intestinal cells marked by Lgr5 function as tissue-resident stem cells that sustain the homeostatic replenishment of the epithelium. By incorporating a diphtheria toxin receptor (DTR) cassette linked to the Lgr5 coding region, native Lgr5-expressing cells are susceptible to ablation upon DT administration in vivo. A similar strategy can be used for Lgr5-expressing cells within organoids established from DTR models. Together, these in vivo and in vitro approaches will facilitate dissection of the roles of Lgr5-expressing cells residing in different tissue compartments. For complete details on the use and execution of this protocol, please refer to Tan et al. (2021).
Gerling M, Büller NV, Kirn LM, Joost S, Frings O, Englert B, Bergström Å, Kuiper RV, Blaas L, Wielenga MC, Almer S, Kühl AA, Fredlund E, van den Brink GR, Toftgård R.
PMID: 27492255 | DOI: 10.1038/ncomms12321
A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.
Leushacke M, Tan SH, Wong A, Swathi Y, Hajamohideen A, Tan LT, Goh J, Wong E, Denil SLIJ, Murakami K, Barker N.
PMID: 28581476 | DOI: 10.1038/ncb3541
The daily renewal of the corpus epithelium is fuelled by adult stem cells residing within tubular glands, but the identity of these stem cells remains controversial. Lgr5 marks homeostatic stem cells and 'reserve' stem cells in multiple tissues. Here, we report Lgr5 expression in a subpopulation of chief cells in mouse and human corpus glands. Using a non-variegated Lgr5-2A-CreERT2 mouse model, we show by lineage tracing that Lgr5-expressing chief cells do not behave as corpus stem cells during homeostasis, but are recruited to function as stem cells to effect epithelial renewal following injury by activating Wnt signalling. Ablation of Lgr5+ cells severely impairs epithelial homeostasis in the corpus, indicating an essential role for these Lgr5+ cells in maintaining the homeostatic stem cell pool. We additionally define Lgr5+ chief cells as a major cell-of-origin of gastric cancer. These findings reveal clinically relevant insights into homeostasis, repair and cancer in the corpus.
Biochemical and Biophysical Research Communications
Choi Y, Park J, Ko YS, Kim Y, Pyo JS, Jange BG, Kim MA, Leef JS, Chang MS, Lee BL.
PMID: - | DOI: 10.1016/j.bbrc.2017.09.163
Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.
L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer
Karuna Ganesh, Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney, Lan He, Roshan Sharma, Kevin P. O�Rourke, Vincent P. Reuter, Yun-Han Huang, Mesruh Turkekul, Ekrem Emrah Er, Ignas Masilionis, Katia Manova-Todorova, Martin R. Weiser, Leonard B. Saltz, Julio Garcia-Aguilar, Richard Koche, Scott W. Lowe, Dana Pe�er, Jinru Shia & Joan Massagu�
| DOI: 10.1038/s43018-019-0006-x
Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin�REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.
