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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.

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IEC18 cell monolayer restitution in response to wounding is dependent on PI3K signaling.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitor of PI3K activation Ly294002 (Ly, 25 µM) or solvent control, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. In a separate set of experiments, the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l, pretreatment of an additional 30 minutes before exposure to Ly) was used to inhibit GSK3ß kinase activity in Ly294002-treated cells. Ly294002 significantly inhibited wounding-induced restitution as compared to control-treated cells, which was not reversed by LiCl-treatment. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
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pone-0026340-g005: IEC18 cell monolayer restitution in response to wounding is dependent on PI3K signaling.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitor of PI3K activation Ly294002 (Ly, 25 µM) or solvent control, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. In a separate set of experiments, the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l, pretreatment of an additional 30 minutes before exposure to Ly) was used to inhibit GSK3ß kinase activity in Ly294002-treated cells. Ly294002 significantly inhibited wounding-induced restitution as compared to control-treated cells, which was not reversed by LiCl-treatment. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).

Mentions: GSK3ß phosphorylation has been implicated in cell spreading and cell migration in various cellular systems [13], [14],[35],[36]. We next determined the role of PI3K activation in wounding-induced IEC18 cell restitution. Of note, Ly294002 exposure significantly decreased wounding-induced cell restitution in IEC18 cells (Fig. 5). Remarkably, adding the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l) to wounded cell monolayers was not able not reverse the effect of Ly294002 treatment (Fig. 5). These findings suggest that PI3K signaling is involved in wounding-induced cellular restitution in IEC18 intestinal epithelial cells. However, the effect of Ly294002 treatment seems to be independent of GSK3ß activity.


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)

IEC18 cell monolayer restitution in response to wounding is dependent on PI3K signaling.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitor of PI3K activation Ly294002 (Ly, 25 µM) or solvent control, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. In a separate set of experiments, the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l, pretreatment of an additional 30 minutes before exposure to Ly) was used to inhibit GSK3ß kinase activity in Ly294002-treated cells. Ly294002 significantly inhibited wounding-induced restitution as compared to control-treated cells, which was not reversed by LiCl-treatment. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198390&req=5

pone-0026340-g005: IEC18 cell monolayer restitution in response to wounding is dependent on PI3K signaling.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitor of PI3K activation Ly294002 (Ly, 25 µM) or solvent control, standardized wounds were created using a razor blade and restitution was monitored over 24 h using serial microphotography. In a separate set of experiments, the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l, pretreatment of an additional 30 minutes before exposure to Ly) was used to inhibit GSK3ß kinase activity in Ly294002-treated cells. Ly294002 significantly inhibited wounding-induced restitution as compared to control-treated cells, which was not reversed by LiCl-treatment. Data are expressed as median and interquartile range (IQR) of n = 9 independent wounds per condition. Statistical analysis was performed by the Kruskal-Wallis test for non-parametric data (*p<0.05 versus control).
Mentions: GSK3ß phosphorylation has been implicated in cell spreading and cell migration in various cellular systems [13], [14],[35],[36]. We next determined the role of PI3K activation in wounding-induced IEC18 cell restitution. Of note, Ly294002 exposure significantly decreased wounding-induced cell restitution in IEC18 cells (Fig. 5). Remarkably, adding the GSK3ß inhibitor Lithium Chloride (LiCl, 20 mmol/l) to wounded cell monolayers was not able not reverse the effect of Ly294002 treatment (Fig. 5). These findings suggest that PI3K signaling is involved in wounding-induced cellular restitution in IEC18 intestinal epithelial cells. However, the effect of Ly294002 treatment seems to be independent of GSK3ß activity.

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