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

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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.

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ATM activation depends on Dclk1 expression and interaction to maintain DNA integrity and cell survival.(a) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions and 48 h post-radiation (4 Gy) were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to ATM. Lower bands represent IgG heavy and short chains. (b) Colony formation assay: We assessed the colony-forming ability of control, vector control, and Dclk1-overexpressing YAMC cells at baseline and 48 h post-radiation (4 Gy). (c) Bar graph represents the percent colony formation of control, vector control, and Dclk1-overexpressing YAMC cells 48 h post-radiation, compared with these cells at baseline. (d) DNA was isolated from YAMC cells of representative groups for an agarose gel DNA fragmentation assay. 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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f8: ATM activation depends on Dclk1 expression and interaction to maintain DNA integrity and cell survival.(a) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions and 48 h post-radiation (4 Gy) were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to ATM. Lower bands represent IgG heavy and short chains. (b) Colony formation assay: We assessed the colony-forming ability of control, vector control, and Dclk1-overexpressing YAMC cells at baseline and 48 h post-radiation (4 Gy). (c) Bar graph represents the percent colony formation of control, vector control, and Dclk1-overexpressing YAMC cells 48 h post-radiation, compared with these cells at baseline. (d) DNA was isolated from YAMC cells of representative groups for an agarose gel DNA fragmentation assay. 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: To test whether the ATM phosphorylation level is directly associated with Dclk1 levels after injury, Dclk1-overexpressing YAMC cells and vector control cells were exposed to radiation and utilized for CO-IP (Fig. 8a). The interaction of ATM and Dclk1 was significantly enhanced after radiation-induced injury. CO-IP revealed that ATM phosphorylation might be directly associated with Dclk1 expression levels after injury. To further test the involvement of Dclk1 in DDR for cell radioprotection and survival/viability, we conducted a colony formation assay post irradiation injury, and found that Dclk1-overexpressing YAMC cells form the same number of colonies as do complete control YAMC cells (not exposed to irradiation), demonstrating that Dclk1-overexpressing cells display enhanced radioprotection after radiation injury (Fig. 8b,c). To examine whether the Dclk1 level is distinct for DNA integrity after injury, Dclk1-overexpressing YAMC cells and vector control cells were exposed to radiation and utilized for a DNA fragmentation assay (Fig. 8d). Compared with vector control cells exposed to irradiation, Dclk1-overexpressing YAMC cells had less DNA damage.


Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury
ATM activation depends on Dclk1 expression and interaction to maintain DNA integrity and cell survival.(a) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions and 48 h post-radiation (4 Gy) were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to ATM. Lower bands represent IgG heavy and short chains. (b) Colony formation assay: We assessed the colony-forming ability of control, vector control, and Dclk1-overexpressing YAMC cells at baseline and 48 h post-radiation (4 Gy). (c) Bar graph represents the percent colony formation of control, vector control, and Dclk1-overexpressing YAMC cells 48 h post-radiation, compared with these cells at baseline. (d) DNA was isolated from YAMC cells of representative groups for an agarose gel DNA fragmentation assay. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f8: ATM activation depends on Dclk1 expression and interaction to maintain DNA integrity and cell survival.(a) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions and 48 h post-radiation (4 Gy) were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to ATM. Lower bands represent IgG heavy and short chains. (b) Colony formation assay: We assessed the colony-forming ability of control, vector control, and Dclk1-overexpressing YAMC cells at baseline and 48 h post-radiation (4 Gy). (c) Bar graph represents the percent colony formation of control, vector control, and Dclk1-overexpressing YAMC cells 48 h post-radiation, compared with these cells at baseline. (d) DNA was isolated from YAMC cells of representative groups for an agarose gel DNA fragmentation assay. 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: To test whether the ATM phosphorylation level is directly associated with Dclk1 levels after injury, Dclk1-overexpressing YAMC cells and vector control cells were exposed to radiation and utilized for CO-IP (Fig. 8a). The interaction of ATM and Dclk1 was significantly enhanced after radiation-induced injury. CO-IP revealed that ATM phosphorylation might be directly associated with Dclk1 expression levels after injury. To further test the involvement of Dclk1 in DDR for cell radioprotection and survival/viability, we conducted a colony formation assay post irradiation injury, and found that Dclk1-overexpressing YAMC cells form the same number of colonies as do complete control YAMC cells (not exposed to irradiation), demonstrating that Dclk1-overexpressing cells display enhanced radioprotection after radiation injury (Fig. 8b,c). To examine whether the Dclk1 level is distinct for DNA integrity after injury, Dclk1-overexpressing YAMC cells and vector control cells were exposed to radiation and utilized for a DNA fragmentation assay (Fig. 8d). Compared with vector control cells exposed to irradiation, Dclk1-overexpressing YAMC cells had less DNA damage.

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