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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.


Related in: MedlinePlus

Dclk1 expressing tuft cells are required to enhance crypt epithelial stemness 24 h post-TBI.(a) Enteroid formation assay: IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI were directly embedded in 0.3% soft agar. (b) Bar graph represents the average number of enteroids formed from IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (c) Western blot analysis of protein expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f 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 = * and < 0.01 = **, 0.001 = *** were considered statistically significant.
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f2: Dclk1 expressing tuft cells are required to enhance crypt epithelial stemness 24 h post-TBI.(a) Enteroid formation assay: IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI were directly embedded in 0.3% soft agar. (b) Bar graph represents the average number of enteroids formed from IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (c) Western blot analysis of protein expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f 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 = * and < 0.01 = **, 0.001 = *** were considered statistically significant.

Mentions: Pluripotency is a central, well-defined feature of stem cells. Analysis of the pluripotency factors expressed in IECs and their self-renewal ability during the injury response may reveal the coordinated molecular pathways that regulate injury/wound repair3233. To understand how Dclk1 expression in the tuft cells contribute to IECs self-renewal and pluripotency at 24 h post-TBI, we investigated the enterosphere-forming ability and expression of pluripotency factors. IECs isolated from VillinCre;Dclk1f/f mice formed 5-fold fewer enterospheres than Dclk1f/f mice (p < 0.0001; Fig. 2a,b). Before TBI, significantly fewer enterospheres were formed in VillinCre;Dclk1f/f mice than in Dclk1f/f mice (p < 0.01; Fig. 2a,b). Thus, the loss of Dclk1 expression in tuft cells reduced the clonogenic capacity of resident crypt epithelial cells, suggesting the paracrine function of tuft cells prerequisite Dclk1 expression to maintain the self-renewal ability of IECs. Before TBI, expression of pluripotency factors Sox2 and Myc were significantly lower in IECs from VillinCre;Dclk1f/f mice than Dclk1f/f mice (p < 0.001; Fig. 2c,d). This finding may represent an additional mechanism of reduced self-renewal during homeostasis. However, 24 h post-TBI, the mRNA and protein levels of Nanog and Sox2 (p < 0.001), and Oct4, Klf4, and Myc (p < 0.0001) were significantly downregulated in IECs from VillinCre;Dclk1f/f mice compared with Dclk1f/f mice (Fig. 2c,d). These results suggest that Dclk1 expression in tuft cells may be required for the paracrine maintenance of intestinal epithelial pluripotency and/or the regeneration of sufficient pluripotent cells to participate in epithelial restitution. However, the secretory factors responsible for the paracrine function of Dclk1 expressing tuft cells need further investigation.


Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury
Dclk1 expressing tuft cells are required to enhance crypt epithelial stemness 24 h post-TBI.(a) Enteroid formation assay: IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI were directly embedded in 0.3% soft agar. (b) Bar graph represents the average number of enteroids formed from IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (c) Western blot analysis of protein expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f 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 = * and < 0.01 = **, 0.001 = *** were considered statistically significant.
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Related In: Results  -  Collection

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f2: Dclk1 expressing tuft cells are required to enhance crypt epithelial stemness 24 h post-TBI.(a) Enteroid formation assay: IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI were directly embedded in 0.3% soft agar. (b) Bar graph represents the average number of enteroids formed from IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (c) Western blot analysis of protein expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f and Dclk1f/f mice, before and 24 h after TBI. (d) RT-PCR analysis of mRNA expression of pluripotency factors Klf4, Nanog, Oct4, Sox2, and cMyc in IECs isolated from VillinCre;Dclk1f/f 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 = * and < 0.01 = **, 0.001 = *** were considered statistically significant.
Mentions: Pluripotency is a central, well-defined feature of stem cells. Analysis of the pluripotency factors expressed in IECs and their self-renewal ability during the injury response may reveal the coordinated molecular pathways that regulate injury/wound repair3233. To understand how Dclk1 expression in the tuft cells contribute to IECs self-renewal and pluripotency at 24 h post-TBI, we investigated the enterosphere-forming ability and expression of pluripotency factors. IECs isolated from VillinCre;Dclk1f/f mice formed 5-fold fewer enterospheres than Dclk1f/f mice (p < 0.0001; Fig. 2a,b). Before TBI, significantly fewer enterospheres were formed in VillinCre;Dclk1f/f mice than in Dclk1f/f mice (p < 0.01; Fig. 2a,b). Thus, the loss of Dclk1 expression in tuft cells reduced the clonogenic capacity of resident crypt epithelial cells, suggesting the paracrine function of tuft cells prerequisite Dclk1 expression to maintain the self-renewal ability of IECs. Before TBI, expression of pluripotency factors Sox2 and Myc were significantly lower in IECs from VillinCre;Dclk1f/f mice than Dclk1f/f mice (p < 0.001; Fig. 2c,d). This finding may represent an additional mechanism of reduced self-renewal during homeostasis. However, 24 h post-TBI, the mRNA and protein levels of Nanog and Sox2 (p < 0.001), and Oct4, Klf4, and Myc (p < 0.0001) were significantly downregulated in IECs from VillinCre;Dclk1f/f mice compared with Dclk1f/f mice (Fig. 2c,d). These results suggest that Dclk1 expression in tuft cells may be required for the paracrine maintenance of intestinal epithelial pluripotency and/or the regeneration of sufficient pluripotent cells to participate in epithelial restitution. However, the secretory factors responsible for the paracrine function of Dclk1 expressing tuft cells need further investigation.

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