Limits...
PI3K-dependent GSK3ß(Ser9)-phosphorylation is implicated in the intestinal epithelial cell wound-healing response.

Karrasch T, Spaeth T, Allard B, Jobin C - PLoS ONE (2011)

Bottom Line: GSK3ß(-/-) cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells.We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response.Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. thomas.karrasch@klinik.uni-regensburg.de

ABSTRACT

Introduction: The ability of the intestinal epithelial barrier to respond to various injurious insults is an essential component of intestinal homeostasis. However, the molecular mechanisms responsible for wound-healing and repair in the intestine are poorly understood. The glycogen synthase kinase 3ß (GSK3ß) has been implicated in various biological processes such as cellular motility, cell spreading and recently inflammation.

Aim: To investigate the role of GSK3ß in intestinal epithelial cell restitution.

Methods: Rat intestinal epithelial IEC18 cells were serum-starved for 16 to 24 h and wounded by multiple scraping. Akt(Ser473)-, GSK3ß(Ser9)- and RelA(Ser536)-phosphorylation were determined by Western blot using specific phospho-antibodies. The inhibitors AG1478 (1 µM) and Ly294002 (25 µM) were used to block EGF-R autophosphorylation and PI3K-activation, respectively. ß-Catenin/LEF/TCF dependent transcription was determined by reporter gene assay (TOP/FOP system). C-myc gene expression was evaluated by real-time RT-PCR. GSK3ß(-/-) mouse embryonic fibroblasts were used to characterize the role of GSK3ß in wounding-induced cell migration.

Results: Wounding induced GSK3ß(Ser9) phosphorylation in IEC-18 cells, which led to ß-catenin accumulation as well as nuclear translocation of ß-catenin. ß-Catenin stabilization/nuclear translocation led to enhanced LEF-TCF transcriptional activity and subsequent c-myc mRNA accumulation in wounded cell monolayers. Blocking PI3K/Akt signaling with Ly294002 prevented wound-induced GSK3ß(Ser9) phosphorylation as well as ß-catenin nuclear translocation and significantly attenuated restitution. Additionally, wounding induced rapid NF-kB(Ser536) phosphorylation, which was inhibited by AG1478, but not by Ly294002. GSK3ß(-/-) cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells.

Conclusion: We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response. Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.

Show MeSH

Related in: MedlinePlus

Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers.Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198390&req=5

pone-0026340-g003: Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers.Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).

Mentions: C-myc gene expression has been characterized as a down-stream target of ß-catenin signal transduction, increasing intestinal epithelial cell survival and proliferation [31], [32]. Thus, to determine the functional relevance of increased TCF/LEF-dependent luciferase activity we measured c-myc mRNA in wounded IEC18 cell monolayers. Interestingly, scrape-wounding induced significant accumulation of c-myc mRNA in IEC18 cells (Fig. 3). In summary, scrape-wounding induces TCF/LEF-dependent gene transcription as well as c-myc mRNA accumulation in confluent intestinal epithelial cell monolayers.


PI3K-dependent GSK3ß(Ser9)-phosphorylation is implicated in the intestinal epithelial cell wound-healing response.

Karrasch T, Spaeth T, Allard B, Jobin C - PLoS ONE (2011)

Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers.Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198390&req=5

pone-0026340-g003: Wounding induces c-myc mRNA accumulation in IEC18 cell monolayers.Rat intestinal epithelial IEC18 cells were grown to confluency in 6-well-plates, serum-starved overnight, and cell monolayers were then wounded by multiple scraping. At the indicated time points, total cell RNA extracts were prepared and analysed for c-myc mRNA accumulation normalized to GAPDH control via real-time RT-PCR using specific primers. mRNA accumulation relative to unwounded control is expressed as median and interquartile range (IQR) of n = 3 separate experiments, each with three separate wells per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
Mentions: C-myc gene expression has been characterized as a down-stream target of ß-catenin signal transduction, increasing intestinal epithelial cell survival and proliferation [31], [32]. Thus, to determine the functional relevance of increased TCF/LEF-dependent luciferase activity we measured c-myc mRNA in wounded IEC18 cell monolayers. Interestingly, scrape-wounding induced significant accumulation of c-myc mRNA in IEC18 cells (Fig. 3). In summary, scrape-wounding induces TCF/LEF-dependent gene transcription as well as c-myc mRNA accumulation in confluent intestinal epithelial cell monolayers.

Bottom Line: GSK3ß(-/-) cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells.We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response.Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. thomas.karrasch@klinik.uni-regensburg.de

ABSTRACT

Introduction: The ability of the intestinal epithelial barrier to respond to various injurious insults is an essential component of intestinal homeostasis. However, the molecular mechanisms responsible for wound-healing and repair in the intestine are poorly understood. The glycogen synthase kinase 3ß (GSK3ß) has been implicated in various biological processes such as cellular motility, cell spreading and recently inflammation.

Aim: To investigate the role of GSK3ß in intestinal epithelial cell restitution.

Methods: Rat intestinal epithelial IEC18 cells were serum-starved for 16 to 24 h and wounded by multiple scraping. Akt(Ser473)-, GSK3ß(Ser9)- and RelA(Ser536)-phosphorylation were determined by Western blot using specific phospho-antibodies. The inhibitors AG1478 (1 µM) and Ly294002 (25 µM) were used to block EGF-R autophosphorylation and PI3K-activation, respectively. ß-Catenin/LEF/TCF dependent transcription was determined by reporter gene assay (TOP/FOP system). C-myc gene expression was evaluated by real-time RT-PCR. GSK3ß(-/-) mouse embryonic fibroblasts were used to characterize the role of GSK3ß in wounding-induced cell migration.

Results: Wounding induced GSK3ß(Ser9) phosphorylation in IEC-18 cells, which led to ß-catenin accumulation as well as nuclear translocation of ß-catenin. ß-Catenin stabilization/nuclear translocation led to enhanced LEF-TCF transcriptional activity and subsequent c-myc mRNA accumulation in wounded cell monolayers. Blocking PI3K/Akt signaling with Ly294002 prevented wound-induced GSK3ß(Ser9) phosphorylation as well as ß-catenin nuclear translocation and significantly attenuated restitution. Additionally, wounding induced rapid NF-kB(Ser536) phosphorylation, which was inhibited by AG1478, but not by Ly294002. GSK3ß(-/-) cells demonstrated significantly attenuated wound-induced restitution compared to wild-type cells.

Conclusion: We conclude that PI3K-mediated GSK3ß phosphorylation is involved in the intestinal epithelial wound-healing response. Phosphorylation of GSK3ß may be important for intestinal restitution by promoting cell motility in response to wounding.

Show MeSH
Related in: MedlinePlus