Mo1065 - P21 Waf1/Cip1 and RNA Binding Protein Musashi-1 Regulate small Intestinal Epithelium Regeneration after Γ Radiation-Induced Injury by Activation of the Subpopulation of Reserve Intestinal Stem Cells In Vivo

2018 ◽  
Vol 154 (6) ◽  
pp. S-688 ◽  
Author(s):  
Emilia J. Orzechowska ◽  
Shestruma Parajuli ◽  
Agnieszka B. Bialkowska ◽  
Vincent W. Yang
Keyword(s):  
Stem Cells ◽  
Intestinal Epithelium ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Intestinal Stem Cells ◽  
Γ Radiation ◽  
Radiation Induced ◽  
Small Intestinal ◽  
Epithelium Regeneration

scholarly journals Interplay among p21Waf1/Cip1, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Emilia J. Orzechowska ◽  
Takahito Katano ◽  
Agnieszka B. Bialkowska ◽  
Vincent W. Yang
Keyword(s):  
Stem Cells ◽  
Gamma Radiation ◽  
Therapeutic Potential ◽  
Mrna Translation ◽  
Negative Regulator ◽  
Intestinal Stem Cells ◽  
Quiescent State ◽  
Γ Radiation ◽  
Regenerative Response ◽  
Radiation Induced

Abstract Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

scholarly journals Delayed activation of the DNA replication licensing system in Lgr5(+) intestinal stem cells

2017 ◽  
Author(s):  
T.D. Carroll ◽  
I.P. Newton ◽  
Y. Chen ◽  
J.J. Blow ◽  
I. Näthke
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Dna Replication ◽  
Intestinal Epithelium ◽  
Intestinal Stem Cells ◽  
Temporal Window ◽  
Restriction Point ◽  
Interphase Cells ◽  
Small Intestinal ◽  
Functional Restriction

ABSTRACTDuring late mitosis and early G1, replication origins are licensed for replication by binding to double hexamers of MCM2-7. Here, we investigate how licensing and proliferative commitment are coupled in the small-intestinal epithelium. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5(+) stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem-cells is caused by an increased G1 length, characterised by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost In Apc mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G1 entry. We propose that the unlicensed G1 of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.

scholarly journals DOT1L-Mediated H3K79 Methylation in Chromatin Is Dispensable for Wnt Pathway-Specific and Other Intestinal Epithelial Functions

2013 ◽  
Vol 33 (9) ◽  
pp. 1735-1745 ◽  
Author(s):  
Li-Lun Ho ◽  
Amit Sinha ◽  
Michael Verzi ◽  
Kathrin M. Bernt ◽  
Scott A. Armstrong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Intestinal Epithelium ◽  
Target Genes ◽  
Intestinal Stem Cells ◽  
Intestinal Epithelial ◽  
Aberrant Expression ◽  
Genome Wide ◽  
Transcript Profiles ◽  
H3k79 Methylation

Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5 + mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se . Selective Dot1l disruption in Lgr5 + stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l -null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo .

scholarly journals High-throughput organoid screening enables engineering of intestinal epithelial composition

2020 ◽  
Author(s):  
Benjamin E. Mead ◽  
Kazuki Hattori ◽  
Lauren Levy ◽  
Marko Vukovic ◽  
Daphne Sze ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
High Throughput ◽  
Intestinal Epithelium ◽  
Paneth Cell ◽  
Stem Cell Differentiation ◽  
Biological Targets ◽  
Small Intestinal

SummaryBarrier tissue epithelia play an essential role in maintaining organismal homeostasis, and changes in their cellular composition have been observed in multiple human diseases. Within the small intestinal epithelium, adult stem cells integrate diverse signals to regulate regeneration and differentiation, thereby establishing overall cellularity. Accordingly, directing stem cell differentiation could provide a tractable approach to alter the abundance or quality of specialized cells of the small intestinal epithelium, including the secretory Paneth, goblet, and enteroendocrine populations. Yet, to date, there has been a lack of suitable tools and rigorous approaches to identify biological targets and pharmacological agents that can modify epithelial composition to enable causal testing of disease-associated changes with novel therapeutic candidates. To empower the search for epithelia-modifying agents, we establish a first-of-its-kind high-throughput phenotypic organoid screen. We demonstrate the ability to screen thousands of samples and uncover biological targets and associated small molecule inhibitors which translate to in vivo. This approach is enabled by employing a functional, cell-type specific, scalable assay on an organoid model designed to represent the physiological cues of in vivo Paneth cell differentiation from adult intestinal stem cells. Further, we miniaturize and adapt the organoid culture system to enable automated plating and screening, thereby providing the ability to test thousands of samples. Strikingly, in our screen we identify inhibitors of the nuclear exporter Xpo1 modulate stem cell fate commitment by inducing a pan-epithelial stress response combined with an interruption of mitogen signaling in cycling intestinal progenitors, thereby significantly increasing the abundance of Paneth cells independent of known WNT and Notch differentiation cues. We extend our observation in vivo, demonstrating that oral administration of Xpo1 inhibitor KPT-330 at doses 1,000-fold lower than conventionally used in hematologic malignancies increases Paneth cell abundance. In total, we provide a framework to identify novel biological cues and therapeutic leads to rebalance intestinal stem cell differentiation and modulate epithelial tissue composition via high-throughput phenotypic screening in rationally-designed organoid model of differentiation.

scholarly journals Krüppel-like factor 4 is a radioprotective factor for the intestine following γ-radiation-induced gut injury in mice

2015 ◽  
Vol 308 (2) ◽  
pp. G121-G138 ◽  
Author(s):  
Daniel Talmasov ◽  
Xinjun Zhang ◽  
Bing Yu ◽  
Mandayam O. Nandan ◽  
Agnieszka B. Bialkowska ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Morphological Changes ◽  
Γ Irradiation ◽  
Γ Radiation ◽  
Proliferative Zone ◽  
Acute Response ◽  
Radiation Induced ◽  
Total Body

Gut radiation-induced injury is a concern during treatment of patients with cancer. Krüppel-like factor 4 (KLF4) is expressed in differentiated villous epithelial cells of the small intestine. We previously showed that KLF4 protects cells from apoptosis following γ-irradiation in vitro. We sought to determine whether KLF4 mediates the small intestinal response to γ-irradiation in vivo. Mice with intestinal epithelium-specific deletion of Klf4 ( Klf4 ΔIS) and control ( Klf4 fl/fl) mice were irradiated with total-body γ-radiation. Following irradiation, the Klf4 ΔIS mice had significantly increased mortality compared with irradiated Klf4 fl/fl mice. Immunohistochemistry and immunofluorescence staining were used to assess the morphological changes, levels of proliferation, and apoptosis in the intestinal epithelium. At 96 h following irradiation, there was a regenerative response manifested by an expansion of the proliferative zone in both mouse groups, with the control mice having a higher proliferative activity than the Klf4 ΔIS group. In addition, there was a significant increase in the number of Klf4/Ki67-copositive cells in the irradiated control mice compared with unirradiated mice. Also, the irradiated Klf4 ΔIS mice had a significantly higher number of crypt cells positive for apoptosis, p53, and p21 compared with irradiated Klf4 fl/fl mice. Taken together, our data suggest that Klf4 may function as a radioprotective factor against gastrointestinal syndrome in mice following γ-irradiation by inhibiting apoptosis in the acute response to irradiation and contributing to crypt regeneration.

scholarly journals A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation

2014 ◽  
Vol 55 (2) ◽  
pp. 381-390 ◽  
Author(s):  
Motohiro Yamauchi ◽  
Kensuke Otsuka ◽  
Hisayoshi Kondo ◽  
Nobuyuki Hamada ◽  
Masanori Tomita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ionizing Radiation ◽  
Intestinal Stem Cells ◽  
Survival Assay ◽  
Small Intestinal ◽  
In Vitro Survival

scholarly journals Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells

Cell Reports ◽  
2021 ◽  
Vol 35 (9) ◽  
pp. 109198
Author(s):  
Shlomi Dvir ◽  
Amir Argoetti ◽  
Chen Lesnik ◽  
Mark Roytblat ◽  
Kohava Shriki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Embryonic Stem

Melatonin attenuates radiation-induced cortical bone-derived stem cells injury and enhances bone repair in postradiation femoral defect model

2021 ◽  
Author(s):  
Wei Hu ◽  
Jiawu Liang ◽  
Song Liao ◽  
Zhidong Zhao ◽  
Yuxing Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Genomic Stability ◽  
Bone Defects ◽  
Self Renewal ◽  
Tnf Α ◽  
Radiation Induced ◽  
Γ Ray

Abstract Background Ionizing radiation poses a challenge to the healing of bone defects. Radiation therapy and accidental exposure to gamma-ray (γ-ray) radiation inhibit bone formation and increase the risk of fractures. Cortical bone-derived stem cells (CBSCs) are essential for osteogenic lineages, bone maintenance, and repair. This study aimed to investigate the effects of melatonin on postradiation CBSCs and bone defects. Methods CBSCs were extracted from C57/BL6 mice and were identified by flow cytometry. The effects of exogenous melatonin on the self-renewal and osteogenic capacity of postradiation CBSCs were detected in vitro. The underlying mechanisms in terms of genomic stability, apoptosis and oxidative stress-related signaling were further analyzed by western blotting, flow cytometry and immunofluorescence. Finally, the effects of melatonin on healing in postradiation bone defects were evaluated in vivo by micro-CT and immunohistochemical analysis. Results The radiation-induced reduced self-renewal and osteogenic capacity were partially reversed in postradiation CBSCs treated with melatonin. Melatonin maintained the genomic stability and apoptosis of postradiation CBSCs, and intracellular oxidative stress was decreased significantly while antioxidant-related enzymes were enhanced. Western blotting verified the anti-inflammatory effect of melatonin by downregulating the levels of IL-6 and TNF-α via extracellular regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, distinct from its antioxidant effect via NRF2 signaling. In vivo experiments demonstrated that the newly formed bone in the melatonin plus Matrigel group had higher trabecular bone volume per tissue volume (BV/TV) and bone mineral density (BMD) values, and lower levels of IL-6 and TNF-α than those in the irradiation and the Matrigel groups. Conclusions This study suggested the potential of melatonin to protect CBSCs against γ-ray radiation and to assist the healing of postradiation bone defects.

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