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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

Tuft cells require Dclk1 expression to mediate its paracrine function.(a) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. (b) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. (c) Co-immunoprecipitation: IEC protein extracts from Dclk1f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. (d) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. (e) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. (f) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1+ tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.
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f9: Tuft cells require Dclk1 expression to mediate its paracrine function.(a) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. (b) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. (c) Co-immunoprecipitation: IEC protein extracts from Dclk1f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. (d) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. (e) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. (f) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1+ tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.

Mentions: Finally, we sought to answer how this small population of Dclk1 expressing tuft cells regulates the whole intestinal epithelium during or after injury. From our mouse model, we learned that COX2 signaling, involved in PGE2 synthesis for paracrine regulation of IECs, was reduced in the VillinCre;Dclk1f/f mice compared with Dclk1f/f mice 24 h and 84 h post-TBI. We observed that COX2 expression and Dclk1/COX2 interaction in Dclk1-overexpressing YAMC cells increases, whereas Dclk1/COX2 interaction decreases, in the IECs of VillinCre;Dclk1f/f mice compared with IECs of Dclk1f/f mice 24 h post-TBI (Fig. 9a–c). We found increased levels of PGE2 in the spent medium from Dclk1-overexpressing YAMC cell culture than in that of vector control YAMC cells, both at baseline and after radiation injury (Fig. 9d). Further, we found that PGE2-treated cells showed similar colony-forming abilities to Dclk1-overexpressing YAMC cells after radiation injury (Fig. 9e). Finally, we observed that co-culture of Dclk1-overexpressing YAMC cells rescued the survival/viability of vector control YAMC cells by increasing their colony-forming abilities after radiation injury (Fig. 9e). These findings suggest that the coordinated regulation of IECs by Dclk1 expressing tuft cells during injury may occur through the paracrine action of prostaglandins. This possibility should be investigated further. Together, these data provide evidence (Fig. 9f) that (i) Dclk1-expressing tuft cells are required for coordinated regulation and/or paracrine regulation of IECs’/ISCs’ radio-resistance critical for their survival and regeneration, and (ii) Dclk1 expression regulates IECs/ISCs survival via enhancing DDR during/after injury.


Intestinal tuft cells regulate the ATM mediated DNA Damage response via Dclk1 dependent mechanism for crypt restitution following radiation injury
Tuft cells require Dclk1 expression to mediate its paracrine function.(a) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. (b) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. (c) Co-immunoprecipitation: IEC protein extracts from Dclk1f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. (d) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. (e) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. (f) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1+ tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f9: Tuft cells require Dclk1 expression to mediate its paracrine function.(a) Western blot analysis was performed to identify the protein expression level of COX2 in the vector control and DCLK1-overexpressing YAMC cells. (b) Co-immunoprecipitation: Protein extracts from vector control YAMC cells and Dclk1-overexpressing cells at basal conditions were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to COX2. Lower bands represent IgG heavy and short chains. (c) Co-immunoprecipitation: IEC protein extracts from Dclk1f/f mice at homeostasis and 24 h post-TBI were co-immunoprecipitated with anti-Dclk1 and blotted with antibody to Cox2. Lower bands represent IgG heavy and short chains. (d) PGE2 EIA assay: Cell culture spent media collected and utilized for analyzing PGE2 levels. (e) Bar graph represents the percent colony formation of YAMC cells of vector control, Dclk1-overexpressing, PGE2-treated, and co-culture (YAMC + YAMC Dclk1-overexpression) cells at baseline and 48 h post-radiation. (f) Proposed Mechanism: Tuft cells regulates ATM mediated DDR in response to injury to enhance intestinal epithelial survival and self-renewal for effective restitution and function. In response to radiation-induced injury, Dclk1+ tuft cells enhance COX2 signaling for the paracrine regulation of IECs/ISCs. Dclk1 expression is critical for ATM activation for the DNA DSB repair response. ATM activation significantly controls DNA repair, apoptosis, and the cell cycle. Cell survival and self-renewal depends upon cellular DNA integrity. Dclk1 regulation of the ATM pathway enhances cell survival and self-renewal for effective intestinal regeneration after TBI.
Mentions: Finally, we sought to answer how this small population of Dclk1 expressing tuft cells regulates the whole intestinal epithelium during or after injury. From our mouse model, we learned that COX2 signaling, involved in PGE2 synthesis for paracrine regulation of IECs, was reduced in the VillinCre;Dclk1f/f mice compared with Dclk1f/f mice 24 h and 84 h post-TBI. We observed that COX2 expression and Dclk1/COX2 interaction in Dclk1-overexpressing YAMC cells increases, whereas Dclk1/COX2 interaction decreases, in the IECs of VillinCre;Dclk1f/f mice compared with IECs of Dclk1f/f mice 24 h post-TBI (Fig. 9a–c). We found increased levels of PGE2 in the spent medium from Dclk1-overexpressing YAMC cell culture than in that of vector control YAMC cells, both at baseline and after radiation injury (Fig. 9d). Further, we found that PGE2-treated cells showed similar colony-forming abilities to Dclk1-overexpressing YAMC cells after radiation injury (Fig. 9e). Finally, we observed that co-culture of Dclk1-overexpressing YAMC cells rescued the survival/viability of vector control YAMC cells by increasing their colony-forming abilities after radiation injury (Fig. 9e). These findings suggest that the coordinated regulation of IECs by Dclk1 expressing tuft cells during injury may occur through the paracrine action of prostaglandins. This possibility should be investigated further. Together, these data provide evidence (Fig. 9f) that (i) Dclk1-expressing tuft cells are required for coordinated regulation and/or paracrine regulation of IECs’/ISCs’ radio-resistance critical for their survival and regeneration, and (ii) Dclk1 expression regulates IECs/ISCs survival via enhancing DDR during/after 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