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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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Wounding-induced GSK3ß(Ser9) phosphorylation and ß-catenin nuclear translocation is dependent on PI3K activation.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitors of PI3K activation Ly294002 (25 µM, Ly) and EGF-R autophosphorylation AG1478 (1 µM) or solvent control and then wounded by multiple scraping. (A) Total cell protein extracts were prepared 30 minutes after wounding and analysed via Western blot. Wounding-induced Akt(Ser473)- and GSK3ß(Ser9)-phosphorylation were blocked by Ly294002 as well as reduced by AG1478. As positive control, AG1478 caused inhibition of RelA phosphorylation (indicating NF-kB activation) after wounding is shown [11]. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. PARP and SOD2 were only detected in the nuclear or cytoplasmic protein fractions, respectively. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p<0.05 versus unwounded control, °p<0.05 versus wounded cells).
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pone-0026340-g004: Wounding-induced GSK3ß(Ser9) phosphorylation and ß-catenin nuclear translocation is dependent on PI3K activation.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitors of PI3K activation Ly294002 (25 µM, Ly) and EGF-R autophosphorylation AG1478 (1 µM) or solvent control and then wounded by multiple scraping. (A) Total cell protein extracts were prepared 30 minutes after wounding and analysed via Western blot. Wounding-induced Akt(Ser473)- and GSK3ß(Ser9)-phosphorylation were blocked by Ly294002 as well as reduced by AG1478. As positive control, AG1478 caused inhibition of RelA phosphorylation (indicating NF-kB activation) after wounding is shown [11]. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. PARP and SOD2 were only detected in the nuclear or cytoplasmic protein fractions, respectively. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p<0.05 versus unwounded control, °p<0.05 versus wounded cells).

Mentions: PI3K has been described to induce phosphorylation of Akt at position Ser473, thereby mediating the phosphorylation of GSK3ß at position Ser9. This in turn inactivates GSK3ß kinase activity [33], [34]. Of note, we observed a significant induction of Akt phosphorylation in response to scrape-wounding IEC18 cell monolayers (Fig. 4A). Thus, we investigated the role of PI3K in wounding-induced GSK3ß phosphorylation. Interestingly, pretreatment with the pharmacological PI3K inhibitor Ly294002 abolished wounding-induced Akt(Ser473)- as well as GSK3ß(Ser9)-phosphorylation in IEC18 cell monolayers (Fig. 4A).


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-induced GSK3ß(Ser9) phosphorylation and ß-catenin nuclear translocation is dependent on PI3K activation.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitors of PI3K activation Ly294002 (25 µM, Ly) and EGF-R autophosphorylation AG1478 (1 µM) or solvent control and then wounded by multiple scraping. (A) Total cell protein extracts were prepared 30 minutes after wounding and analysed via Western blot. Wounding-induced Akt(Ser473)- and GSK3ß(Ser9)-phosphorylation were blocked by Ly294002 as well as reduced by AG1478. As positive control, AG1478 caused inhibition of RelA phosphorylation (indicating NF-kB activation) after wounding is shown [11]. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. PARP and SOD2 were only detected in the nuclear or cytoplasmic protein fractions, respectively. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p<0.05 versus unwounded control, °p<0.05 versus wounded cells).
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pone-0026340-g004: Wounding-induced GSK3ß(Ser9) phosphorylation and ß-catenin nuclear translocation is dependent on PI3K activation.Confluent IEC18 cell monolayers were pretreated for 30 minutes with the specific inhibitors of PI3K activation Ly294002 (25 µM, Ly) and EGF-R autophosphorylation AG1478 (1 µM) or solvent control and then wounded by multiple scraping. (A) Total cell protein extracts were prepared 30 minutes after wounding and analysed via Western blot. Wounding-induced Akt(Ser473)- and GSK3ß(Ser9)-phosphorylation were blocked by Ly294002 as well as reduced by AG1478. As positive control, AG1478 caused inhibition of RelA phosphorylation (indicating NF-kB activation) after wounding is shown [11]. (B) 30 minutes after wounding, cells were lysed, subcellular fractions of nuclear/cytoplasmic protein were prepared and analysed for phospho-GSK3ß(Ser9) and ß-catenin accumulation via Western blot. PARP and SOD2 were only detected in the nuclear or cytoplasmic protein fractions, respectively. (A,B) Densitometry was performed on n = 3–4 different Western blots (each representative of an independent experiment) per condition and normalized to the respective loading controls (GAPDH, SOD, PARP). Protein expression is expressed as -fold protein induction relative to the respective unwounded control. Data are expressed as means ± S.D. of 3–4 different experiments per condition. Statistical analysis was performed by the two-tailed Student's t test (*p<0.05 versus unwounded control, °p<0.05 versus wounded cells).
Mentions: PI3K has been described to induce phosphorylation of Akt at position Ser473, thereby mediating the phosphorylation of GSK3ß at position Ser9. This in turn inactivates GSK3ß kinase activity [33], [34]. Of note, we observed a significant induction of Akt phosphorylation in response to scrape-wounding IEC18 cell monolayers (Fig. 4A). Thus, we investigated the role of PI3K in wounding-induced GSK3ß phosphorylation. Interestingly, pretreatment with the pharmacological PI3K inhibitor Ly294002 abolished wounding-induced Akt(Ser473)- as well as GSK3ß(Ser9)-phosphorylation in IEC18 cell monolayers (Fig. 4A).

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