ABSTRACT
BACKGROUND: Growth of the small intestine in the infant rat is promoted by crypt fission and later by increased crypt cell proliferation. Notch signaling could promote crypt fission. Hes-1 is a Notch target gene. AIM: We assessed the effect of Notch signaling on intestinal crypt fission and on growth of the intestine in the infant rat. METHODS: Hes-1 expression was determined in the small intestine of litters of Hooded Wistar rats aged between 3 and 72 days. Hes-1 RNA expression was measured by quantitative RT-PCR. Four groups of rats (n = 8 or 9) were injected daily, ip, either with vehicle or with the Notch inhibitor DAPT at doses of 3, 10, and 30 mg/kg, from days 9 to 13 of life, and killed on day 14. A microdissection technique was used to measure crypt fission, mitotic count, and apoptotic count. Data were analyzed by ANOVA and by use of Dunnett's F test. RESULTS: Hes-1 expression and crypt fission peaked on day 14. DAPT reduced Hes-1 immunostaining in proportion to dose. DAPT reduced villous area to 72 % (p < 0.01), 53 % (p < 0.001), and 38 % (p < 0.001) of control values for 3, 10 and 30 mg/kg doses, respectively, and reduced crypt fission to 53 % (p < 0.001) and 38 % (p < 0.001) of control values, respectively, for 10 and 30 mg/kg doses. Crypt mitotic count was not affected by any DAPT dose. DAPT at 10 and 30 mg/kg significantly increased apoptosis in crypts, by 6.5 and 4.8-fold, respectively. CONCLUSIONS: We conclude that Notch signaling promotes crypt fission and growth of the intestine by maintaining low apoptosis of crypt cells.
Subject(s)
Intestine, Small/growth & development , Intestine, Small/metabolism , Receptors, Notch/metabolism , Signal Transduction/physiology , Aging , Animals , Apoptosis , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA, Complementary/genetics , DNA, Complementary/metabolism , Dipeptides/pharmacology , Female , Gene Expression Regulation, Developmental/physiology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Male , RNA/genetics , RNA/metabolism , Rats , Rats, Wistar , Real-Time Polymerase Chain Reaction , Receptors, Notch/genetics , Transcription Factor HES-1ABSTRACT
OBJECTIVES: Intestinal crypt fission peaks during infancy. In human and experimental familial polyposis coli, increased crypt fission is due to activation of Wnt/ß-catenin signalling, but the molecular basis of crypt fission during intestinal growth has not been examined. The aim of this project was to investigate whether crypt fission and intestinal growth are affected by experimental blockade of the Wnt/ß-catenin signalling pathway. METHODS: Hooded Wistar rats were given either the Wnt inhibitor, dickkopf (30 and 100 ng), daily or vehicle control intraperitoneally from days 11 to 15 and were killed at day 16. Intestinal morphometry was used to measure villous area, crypt area, percentage of crypt fission, and crypt mitotic count. Intestinal stem cells were assessed by expression of real time-polymerase chain reaction for Lgr5 (a stem cell marker), and the number of ß-catenin-expressing crypts by immunostaining was determined after 100-ng dickkopf treatment. RESULTS: Dickkopf at 30 and 100 ng/day reduced villous area to 71% (P = 0.013) and 29% (P < 0.0001), crypt area to 42% (P = 0.0026) and 30% (P = 0.0067), and crypt fission to 51% (P = 0.006) and 29% (P < 0.0001), respectively, of control values. Mitotic count per crypt did not change. Lgr5 RNA expression and the number of ß-catenin-expressing crypts decreased in dickkopf-treated animals. CONCLUSIONS: We conclude that intestinal crypt fission during infancy is mediated by Wnt signalling. It is possible that local treatment with Wnt agonists could be used to increase intestinal growth.
Subject(s)
Intercellular Signaling Peptides and Proteins/pharmacology , Intestinal Mucosa/metabolism , Intestines/drug effects , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism , Animals , Intestines/pathology , Mitotic Index , Polycomb Repressive Complex 1/drug effects , Polycomb Repressive Complex 1/metabolism , RNA/metabolism , Rats , Rats, Wistar , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Stem Cells/metabolism , beta Catenin/drug effectsABSTRACT
The integrity, or barrier function, of the intestinal epithelium is of paramount importance in -maintaining good health. This is largely imparted by a single layer of epithelial cells linked by the transmembrane tight junction protein complex near their apical surface. Disruption of epithelial permeability via the tight junctions can contribute to disease progression. The cytokine IFNγ is involved in many inflammatory processes and has been shown to dramatically increase permeability via changes at the tight junction in experimental models. One of its key effectors is the transcription factor, -IRF-1. In our studies of the role of IRF-1 in barrier function using the human T84 intestinal epithelial cell monolayer model, we have found that induction of IRF-1 alone is insufficient to change permeability and that if IRF-1 is involved in mediating the permeability effects of IFNγ, then other factors must also be required.
