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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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PI3K-dependent GSK3ß(Ser9) phosphorylation is implicated in the intestinal epithelial cell wound-healing response (Schematic representation).(A) In resting intestinal epithelial cell monolayers, PI3K is inactive, and GSK3ß in its unphosphorylated (active) state phosphorylates ß-catenin, leading to its inactivation. (B) Wounding activates PI3K, leading to GSK3ß phosphorylation at position Ser9. This phosphorylation inactivates GSK3ß, leading to ß-catenin accumulation in intestinal epithelial cells. ß-catenin translocates to the nucleus, modulating gene expression, cell survival and potentially cell migration as well.
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pone-0026340-g007: PI3K-dependent GSK3ß(Ser9) phosphorylation is implicated in the intestinal epithelial cell wound-healing response (Schematic representation).(A) In resting intestinal epithelial cell monolayers, PI3K is inactive, and GSK3ß in its unphosphorylated (active) state phosphorylates ß-catenin, leading to its inactivation. (B) Wounding activates PI3K, leading to GSK3ß phosphorylation at position Ser9. This phosphorylation inactivates GSK3ß, leading to ß-catenin accumulation in intestinal epithelial cells. ß-catenin translocates to the nucleus, modulating gene expression, cell survival and potentially cell migration as well.

Mentions: Our data further characterize GSK3ß's role during immediate intestinal epithelial cell restitution: Wounding IEC18 cell monolayers leads to rapid ß-catenin accumulation and nuclear translocation via PI3K-dependent GSK3ß inhibition through its phosphorylation at position Ser 9. This is followed by TCF/LEF-dependent gene expression and accumulation of c-myc mRNA in wounded cells, which has been demonstrated to increase intestinal epithelial cell survival and proliferation [31], [32] (Fig. 7A, B).


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)

PI3K-dependent GSK3ß(Ser9) phosphorylation is implicated in the intestinal epithelial cell wound-healing response (Schematic representation).(A) In resting intestinal epithelial cell monolayers, PI3K is inactive, and GSK3ß in its unphosphorylated (active) state phosphorylates ß-catenin, leading to its inactivation. (B) Wounding activates PI3K, leading to GSK3ß phosphorylation at position Ser9. This phosphorylation inactivates GSK3ß, leading to ß-catenin accumulation in intestinal epithelial cells. ß-catenin translocates to the nucleus, modulating gene expression, cell survival and potentially cell migration as well.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026340-g007: PI3K-dependent GSK3ß(Ser9) phosphorylation is implicated in the intestinal epithelial cell wound-healing response (Schematic representation).(A) In resting intestinal epithelial cell monolayers, PI3K is inactive, and GSK3ß in its unphosphorylated (active) state phosphorylates ß-catenin, leading to its inactivation. (B) Wounding activates PI3K, leading to GSK3ß phosphorylation at position Ser9. This phosphorylation inactivates GSK3ß, leading to ß-catenin accumulation in intestinal epithelial cells. ß-catenin translocates to the nucleus, modulating gene expression, cell survival and potentially cell migration as well.
Mentions: Our data further characterize GSK3ß's role during immediate intestinal epithelial cell restitution: Wounding IEC18 cell monolayers leads to rapid ß-catenin accumulation and nuclear translocation via PI3K-dependent GSK3ß inhibition through its phosphorylation at position Ser 9. This is followed by TCF/LEF-dependent gene expression and accumulation of c-myc mRNA in wounded cells, which has been demonstrated to increase intestinal epithelial cell survival and proliferation [31], [32] (Fig. 7A, B).

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