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Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury

View Article: PubMed Central - PubMed

ABSTRACT

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

No MeSH data available.


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Intestine-specific Dclk1-KO mice exhibit intestinal crypt epithelial hypoplasia and impaired stem cell population 24 h post-TBI.(a,b) Intestinal tissue sections from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and after TBI (24 h), were stained for H&E and anti-Dclk1. (c) Western blot analysis of protein expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and 24 h after TBI. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of< 0.05 = *, <0.01 = ** and 0.001 = *** were considered statistically significant.
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f1: Intestine-specific Dclk1-KO mice exhibit intestinal crypt epithelial hypoplasia and impaired stem cell population 24 h post-TBI.(a,b) Intestinal tissue sections from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and after TBI (24 h), were stained for H&E and anti-Dclk1. (c) Western blot analysis of protein expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and 24 h after TBI. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of< 0.05 = *, <0.01 = ** and 0.001 = *** were considered statistically significant.

Mentions: Dclk1 expression is restricted to tuft cells in the small intestine. To explore the possible functional role of Dclk1 expression in tuft cells regulating the ISC response with respect to crypt epithelial proliferation and differentiation, we subjected VillinCre;Dclk1f/f mice, a model that lacks Dclk1 expression in the whole intestinal epithelial cells (Fig. 1b), and Dclk1f/f mice (wild-type littermates) to 12 Gy lethal TBI to induce severe epithelial injury. In the absence of injury, crypt architecture and intestinal histology were not notably different in VillinCre;Dclk1f/f mice compared with Dclk1f/f mice (Fig. 1a), indicating that Dclk1-expressing tuft cells may be dispensable for crypt homeostasis under normal conditions. We compared the crypt architecture and intestinal histology and DNA damage between the VillinCre mice and Dclk1f/f mice and found no change in phenotype between these mice (Suppl. Fig. 1). At 24 h post-TBI, intestinal crypt architecture and epithelial arrangement in the crypt were deranged, and a reduction in crypt epithelial cell number was observed in Dclk1f/f mice (Fig. 1a)31. VillinCre;Dclk1f/f mice displayed acute crypt hypoplasia, markedly distorted and shortened crypts that become widely separated from the underlying muscularis mucosae, and a dramatic reduction in IECs, indicating that Dclk1 expression is critical in maintaining crypt epithelial morphology and cell number 24 h after severe genotoxic injury (Fig. 1a). The expression of ISC markers Lgr5, Bmi1, and Musashi1 was also markedly reduced in the IECs of VillinCre;Dclk1f/f mice compared with Dclk1f/f mice 24 h post-TBI (Fig. 1c,d), suggesting that Lgr5+, Bmi1+, and Musashi1+ stem cells were reduced in the VillinCre;Dclk1f/f animals. Lgr5 expression was reduced even before TBI, suggesting that the regulation of Lgr5 expression and or Lgr5+ stem cells under homeostatic conditions may require Dclk1 expression in tuft cells in the intestine (Fig. 1c,d). These data suggest that Dclk1 is required in tuft cells to protect and/or maintain crypt epithelial morphology after injury, perhaps it also suggested that possible coordination between Dclk1 expressing tuft cells and additional stem cell proteins or stem cells is prerequisite.


Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury
Intestine-specific Dclk1-KO mice exhibit intestinal crypt epithelial hypoplasia and impaired stem cell population 24 h post-TBI.(a,b) Intestinal tissue sections from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and after TBI (24 h), were stained for H&E and anti-Dclk1. (c) Western blot analysis of protein expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and 24 h after TBI. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of< 0.05 = *, <0.01 = ** and 0.001 = *** were considered statistically significant.
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Related In: Results  -  Collection

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f1: Intestine-specific Dclk1-KO mice exhibit intestinal crypt epithelial hypoplasia and impaired stem cell population 24 h post-TBI.(a,b) Intestinal tissue sections from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and after TBI (24 h), were stained for H&E and anti-Dclk1. (c) Western blot analysis of protein expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of stem cell markers Bmi1, Lgr5, Msi1, and Dclk1 in isolated IECs from VillinCre;Dclk1f/f mice and Dclk1f/f mice, before and 24 h after TBI. All quantitative data are expressed as means ± SD of a minimum of three independent experiments. P values of< 0.05 = *, <0.01 = ** and 0.001 = *** were considered statistically significant.
Mentions: Dclk1 expression is restricted to tuft cells in the small intestine. To explore the possible functional role of Dclk1 expression in tuft cells regulating the ISC response with respect to crypt epithelial proliferation and differentiation, we subjected VillinCre;Dclk1f/f mice, a model that lacks Dclk1 expression in the whole intestinal epithelial cells (Fig. 1b), and Dclk1f/f mice (wild-type littermates) to 12 Gy lethal TBI to induce severe epithelial injury. In the absence of injury, crypt architecture and intestinal histology were not notably different in VillinCre;Dclk1f/f mice compared with Dclk1f/f mice (Fig. 1a), indicating that Dclk1-expressing tuft cells may be dispensable for crypt homeostasis under normal conditions. We compared the crypt architecture and intestinal histology and DNA damage between the VillinCre mice and Dclk1f/f mice and found no change in phenotype between these mice (Suppl. Fig. 1). At 24 h post-TBI, intestinal crypt architecture and epithelial arrangement in the crypt were deranged, and a reduction in crypt epithelial cell number was observed in Dclk1f/f mice (Fig. 1a)31. VillinCre;Dclk1f/f mice displayed acute crypt hypoplasia, markedly distorted and shortened crypts that become widely separated from the underlying muscularis mucosae, and a dramatic reduction in IECs, indicating that Dclk1 expression is critical in maintaining crypt epithelial morphology and cell number 24 h after severe genotoxic injury (Fig. 1a). The expression of ISC markers Lgr5, Bmi1, and Musashi1 was also markedly reduced in the IECs of VillinCre;Dclk1f/f mice compared with Dclk1f/f mice 24 h post-TBI (Fig. 1c,d), suggesting that Lgr5+, Bmi1+, and Musashi1+ stem cells were reduced in the VillinCre;Dclk1f/f animals. Lgr5 expression was reduced even before TBI, suggesting that the regulation of Lgr5 expression and or Lgr5+ stem cells under homeostatic conditions may require Dclk1 expression in tuft cells in the intestine (Fig. 1c,d). These data suggest that Dclk1 is required in tuft cells to protect and/or maintain crypt epithelial morphology after injury, perhaps it also suggested that possible coordination between Dclk1 expressing tuft cells and additional stem cell proteins or stem cells is prerequisite.

View Article: PubMed Central - PubMed

ABSTRACT

Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of VillinCre;Dclk1f/f mice were hypoplastic and more apoptotic 24&thinsp;h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.

No MeSH data available.


Related in: MedlinePlus