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Insights into intestinal regeneration signaling mechanisms.

ABSTRACT: The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/?-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine the potential role of the canonical Wnt pathway on intestine regeneration in the sea cucumber Holothuria glaberrima. We evaluated their effects in vivo by using histological analyses for cell dedifferentiation, cell proliferation and apoptosis. We found that iCRT14, an alleged Wnt pathway inhibitor, decreased the size of the regenerating intestine, while LiCl, a presumed Wnt pathway activator, increased its size. The possible cellular mechanisms by which signaling pathway disruptors affect the gut rudiment size were further studied in vitro, using cultures of tissue explants and additional pharmacological agents. Among the tested signaling activators, those that act through GSK-3 inhibition, LiCl, 1-Azakenpaullone, and CHIR99021 were found to increase muscle cell dedifferentiation, while the inhibitor iCRT14 blocked cell dedifferentiation. Differently, cell proliferation was reduced by all GSK-3 inhibitors, as well as by iCRT14 and C59, which interferes with Wnt ligand secretion. The in vivo temporal and spatial pattern of ?-catenin activity was determined using an antibody against phosphorylated ?-catenin and shown to correlate with cell proliferative activity. In vitro treatment using C59 decreased the number of cells immunostained for nuclear phosphorylated ?-catenin. Our results showed that the cell dedifferentiation observed during intestinal regeneration can be decoupled from the cell proliferation event and that these cellular processes can be modulated by particular signaling pathway inhibitors and activators. These results open the door for future studies where the cellular signaling pathways involved at each regeneration stage can be determined.

SUBMITTER: Bello SA

PROVIDER: S-EPMC6987020 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Insights into intestinal regeneration signaling mechanisms.

Bello Samir A SA Torres-Gutiérrez Vanessa V Rodríguez-Flores Eneric J EJ Toledo-Román Ernesto J EJ Rodríguez Natalia N Díaz-Díaz Lymarie M LM Vázquez-Figueroa Lionel D LD Cuesta José M JM Grillo-Alvarado Valentina V Amador Alexandra A Reyes-Rivera Josean J García-Arrarás José E JE

Developmental biology 20191009 1

The cellular mechanisms underlying the amazing ability of sea cucumbers to regenerate their autotomized intestines have been widely described by us and others. However, the signaling pathways that control these mechanisms are unknown. Previous studies have shown that Wnt homologs are upregulated during early intestinal regenerative stages, suggesting that the Wnt/β-catenin pathway is active during this process. Here, we used small molecules, putative disruptors of the Wnt pathway, to determine t ...[more]

PMID: 31605680

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Project description:Female C57BL/6J mice were fed a vitamin D deficient diet (0.47% calcium, 0.3% phosphate; Teklad diet TD 89123, Envigo Teklad diets, Madison, Wisconsin) 2 -3 weeks prior to mating to a male C57BL/6J male mouse and during subsequent pregnancy and lactation. Pups from these mice were maintained on this diet until 12-14 weeks of age. For this study, mice were maintained in a virus and parasite-free barrier facility and exposed to a 12h-light, 12h-dark cycle. Food and water were given ad libitum. All the animal experiments conducted were approved by the Rutgers, New Jersey Medical School Animal Care and Use Committee. Mice were ordered from Charles River Laboratories. 12 – 14 week old vitamin D deficient mice were randomly separated and injected with either 0.1 ml vehicle (9:1 mix of propylene glycol and ethanol) or 1,25(OH)2D3 (from Cayman Chemical Company Ann Arbor, MI) (ip 10ng/g body weight) and sacrificed 4h after the injection. Intestinal tissues were harvested from mice. About 10-15 cm of the proximal half of small intestine were used for the duodenum crypts and villi samples, and the entire colon tissues from the terminal cecum to rectum were used for colon samples. After flushing with cold PBS, the intestines were cut opened longitudinally, cut into 1 cm pieces in cold PBS for washing, and then incubated with 3mM EDTA/PBS in rotator for 5 min at 4 0C. The solution was discarded and the tissues were incubated with fresh 3mM EDTA/PBS in rotator for 10 min at 4 0C. After shaking the tubes gently for 10 times, the intestinal pieces were transferred to new tube prepared with cold 3mM EDTA/PBS, rotated for 30 min at 4 0C, then were shaken 30 times. The supernatant containing the whole epithelium tissues (crypts and villi) were collected and spun down at 200 rcf for 3 min at 4 0C. The pellet was resuspended with cold PBS and villi separated from crypts with 70 µm filter. Colon tissue was scraped with a glass slide to remove the epithelial mucosa and washed with PBS. All samples were centrifuged tubes at 200 rcf for 3 mins at 4 0C and at 300 rcf for 30 seconds at 40C in order to remove any residual PBS. We then proceeded to RNA extraction. RNeasy Plus Universal Kit was used for villi and crypts with RiboZol RNA extraction reagent (Amresco, Solon, Ohio), according to manufacturer’s instructions. All nucleic acid extracts were gDNA Eliminator Solution for 15 s at 37 °C, in order to remove contaminating chromosomal DNA. Resulting RNA was analyzed for quantity and quality with a NanoDrop spectrophotometer ND-1000 (Isogen Life Science, Utrecht, The Netherlands).

Project description:Background: Acute gastrointestinal syndrome (AGS) is one of the most severe clinical manifestations after exposure to high doses of radiation, and is life-threatening in radiological emergency scenarios. However, an unmet challenge is lacking of an FDA-approved drug that can ameliorate the damage of radiation-exposed intestinal tissues and accelerate the regeneration of injured epithelia. In this study, we investigated whether the small molecule Me6TREN (Me6) can regulate intestinal stem cell (ISC) proliferation and promote crypt regeneration after irradiation. Methods: Lethally irradiated mice were administered with Me6 or PBS to study the survival rate, and sections of their small intestine were subjected to immunostaining to evaluate epithelial regeneration. An intestinal organoid culture system was employed to detect the role of Me6 in organoid growth and ISC proliferation. We further investigated the key signaling pathways associated with Me6 using microarray, western blotting, and RNA interference techniques. Results: We identified the small molecule Me6 as a potent intestinal radiation countermeasure. Systemic administration of Me6 significantly improved ISC and crypt cell regeneration and enhanced the survival of mice after high doses of radiation. Using an in vitro intestinal organoid culture system, we found that Me6 not only induced ISC proliferation but also increased the budding rate of intestinal organoids under unirradiated and irradiated conditions. Me6 remarkably activated the expression of ISC-associated and proliferation-promoting genes, such as Ascl2, Lgr5, Myc, and CyclinD1. Mechanistically, Me6 strongly stimulated the phosphorylation of β-catenin at the S552 site and increased the transcriptional activity of β-catenin, a key signaling pathway for ISC self-renewal and proliferation. This is further evidenced by the fact that knockdown of β-catenin abolished the effect of Me6 on intestinal organoid growth in vitro and crypt regeneration in irradiated mice. Conclusion: The small molecule Me6TREN induced ISC proliferation, enhanced intestinal organoid growth in vitro, and promoted intestinal tissue regeneration after radiation injury by activating β-catenin signaling.

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Insights into intestinal regeneration signaling mechanisms.

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Insights into intestinal regeneration signaling mechanisms.

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