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Activation of PKCβII by PMA facilitates enhanced epithelial wound repair through increased cell spreading and migration.

Sumagin R, Robin AZ, Nusrat A, Parkos CA - PLoS ONE (2013)

Bottom Line: We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours.Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin.These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. ronen.sumagin@emory.edu

ABSTRACT
Rapid repair of epithelial wounds is essential for intestinal homeostasis, and involves cell proliferation and migration, which in turn are mediated by multiple cellular signaling events including PKC activation. PKC isoforms have been implicated in regulating cell proliferation and migration, however, the role of PKCs in intestinal epithelial cell (IEC) wound healing is still not completely understood. In the current work we used phorbol 12-myristate 13-acetate (PMA), a well recognized agonist of classical and non-conventional PKC subfamilies to investigate the effect of PKC activation on IEC wound healing. We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours. The PMA effect was specifically mediated by PKCβII, as its inhibition significantly diminished the PMA-induced increase in wound closure. Furthermore, we show that the PKCβII-mediated increase in IEC wound closure after PMA stimulation was mediated by increased cell spreading/cell migration but not proliferation. Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin. These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.

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PKCβII activation by PMA increased assembly of focal adhesions.Scratch wounded IEC monolayers were allowed to migrate for 4 hours in the absence (control) or presence of PMA (PMA) and PKC isoforms inhibitors. The levels of total and phospo-paxillin were determined by immunoblotting (A) and quantified using densitometric analysis (B). The data are expressed as fold change relative to control after normalization to loading control. PMA treatment increased paxillin phosphorylation, which was attenuated by the inhibition of PKCβII (PMA+βII), but not PKCβI (PMA+βI). (C) Cell adhesion assay was used to examine IEC-matrix adhesion after PMA treatment in the presence or absence of specific inhibitors of PKC isoforms. PMA treatment significantly increased cell-matrix adhesion.This effect was reversed with inhibition of PKCβII, but not PKCβI. *significantly different (p<0.05). (D) To examine formation of focal adhesions by the cells at the leading edge (upper panels), and cells away from the wound (bottom panels) IECs were fixed and stained for F-actin (red) and phospho-paxillin (green). Representative images show increased phosphorylation of paxillin both at the lamellipodium and areas remote from the wound after PKC activation with PMA. The bar is 20 µm. (E–F) Quantification of the changes in the relative fluorescence intensity, as an indication of the changes in paxillin phosphorylation. (E) Average relative fluorescence intensity of 5 randomly placed lines (as shown in (D), bottom panels) per field of view, at least 7 fields per condition. N = 3 independent experiments. **significantly different (p<0.01). (F) Fluorescence intensity profiles of phospho-paxillin along representative white lines projected across fields of view (as shown on the images in (D), bottom panels) that were used for quantification in (E).
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pone-0055775-g006: PKCβII activation by PMA increased assembly of focal adhesions.Scratch wounded IEC monolayers were allowed to migrate for 4 hours in the absence (control) or presence of PMA (PMA) and PKC isoforms inhibitors. The levels of total and phospo-paxillin were determined by immunoblotting (A) and quantified using densitometric analysis (B). The data are expressed as fold change relative to control after normalization to loading control. PMA treatment increased paxillin phosphorylation, which was attenuated by the inhibition of PKCβII (PMA+βII), but not PKCβI (PMA+βI). (C) Cell adhesion assay was used to examine IEC-matrix adhesion after PMA treatment in the presence or absence of specific inhibitors of PKC isoforms. PMA treatment significantly increased cell-matrix adhesion.This effect was reversed with inhibition of PKCβII, but not PKCβI. *significantly different (p<0.05). (D) To examine formation of focal adhesions by the cells at the leading edge (upper panels), and cells away from the wound (bottom panels) IECs were fixed and stained for F-actin (red) and phospho-paxillin (green). Representative images show increased phosphorylation of paxillin both at the lamellipodium and areas remote from the wound after PKC activation with PMA. The bar is 20 µm. (E–F) Quantification of the changes in the relative fluorescence intensity, as an indication of the changes in paxillin phosphorylation. (E) Average relative fluorescence intensity of 5 randomly placed lines (as shown in (D), bottom panels) per field of view, at least 7 fields per condition. N = 3 independent experiments. **significantly different (p<0.01). (F) Fluorescence intensity profiles of phospho-paxillin along representative white lines projected across fields of view (as shown on the images in (D), bottom panels) that were used for quantification in (E).

Mentions: Cellular protrusions at the leading edge have to adhere to the matrix to mediate forward cell movement. Such matrix adhesions in migrating cells are referred to as focal complexes. Paxillin, is a key protein that regulates focal cell matrix adhesion dynamics [9]. Therefore we asked whether PMA stimulation of IECs results in increased activation of paxillin (increased phosphorylation, Y118), and if this event is mediated by PKCβII. Using western blot analysis we found increased phospho-paxillin (∼2.6-fod) 4 hours after PMA stimulation (Fig. 6A,B). A small non-significant increase in total paxillin level (∼1.1-fold) was observed. Inhibition of PKCβII, but not PKCβI prevented the PMA induced increase in paxillin phosphorylation consistent with the effect of PMA and PKCβII inhibition on lamellipodia formation and wound closure. Similar results were observed with another key protein that is activated in focal complexes during cell adhesion and migration, focal adhesion kinse (FAK) [46]. PMA treatment significantly increased FAK phosphorylation (Y397), which was reversed with inhibition of PKCβII, but not PKCβI (Figure S4). As both phospho-paxillin and phospho-FAK were previously reported in engaged cell-matrix adhesions [46], we next investigated the role for PMA and PKCβII in IEC adhesion to extracellular matrix. PMA treatment increased cell-matrix adhesion (∼1.8-fold). This increase was reversed in the presence of PKCβII, but not PKCβI pharmacologic inhibitors (Fig. 6C), confirming that PKCβII activation by PMA enhances assembly of focal adhesions. Furthermore, as shown in representative immunofluorescence images (Fig. 6D upper panels) PMA induced lamellipodia had increased phospho-paxillin (Y118) that was observed in aggregates suggestive of focal adhesion plaques at the leading edge of spreading IECs. Interestingly, PMA treatment also significantly increased phosphorylation of paxillin in IECs located greater than 100 µm away from the wound (Fig. 6D bottom panels, and quantification Fig. 7E,F). Epithelial cells migrate as a sheet, where cells removed from the leadings edge also contribute to wound closure by cell spreading. As paxillin and FAK play an important role in cell spreading, our findings suggest that PMA-induced activation of paxillin in IECs is accompanied by spreading of the entire epithelial sheet and not just cells at the leading edge.


Activation of PKCβII by PMA facilitates enhanced epithelial wound repair through increased cell spreading and migration.

Sumagin R, Robin AZ, Nusrat A, Parkos CA - PLoS ONE (2013)

PKCβII activation by PMA increased assembly of focal adhesions.Scratch wounded IEC monolayers were allowed to migrate for 4 hours in the absence (control) or presence of PMA (PMA) and PKC isoforms inhibitors. The levels of total and phospo-paxillin were determined by immunoblotting (A) and quantified using densitometric analysis (B). The data are expressed as fold change relative to control after normalization to loading control. PMA treatment increased paxillin phosphorylation, which was attenuated by the inhibition of PKCβII (PMA+βII), but not PKCβI (PMA+βI). (C) Cell adhesion assay was used to examine IEC-matrix adhesion after PMA treatment in the presence or absence of specific inhibitors of PKC isoforms. PMA treatment significantly increased cell-matrix adhesion.This effect was reversed with inhibition of PKCβII, but not PKCβI. *significantly different (p<0.05). (D) To examine formation of focal adhesions by the cells at the leading edge (upper panels), and cells away from the wound (bottom panels) IECs were fixed and stained for F-actin (red) and phospho-paxillin (green). Representative images show increased phosphorylation of paxillin both at the lamellipodium and areas remote from the wound after PKC activation with PMA. The bar is 20 µm. (E–F) Quantification of the changes in the relative fluorescence intensity, as an indication of the changes in paxillin phosphorylation. (E) Average relative fluorescence intensity of 5 randomly placed lines (as shown in (D), bottom panels) per field of view, at least 7 fields per condition. N = 3 independent experiments. **significantly different (p<0.01). (F) Fluorescence intensity profiles of phospho-paxillin along representative white lines projected across fields of view (as shown on the images in (D), bottom panels) that were used for quantification in (E).
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Related In: Results  -  Collection

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pone-0055775-g006: PKCβII activation by PMA increased assembly of focal adhesions.Scratch wounded IEC monolayers were allowed to migrate for 4 hours in the absence (control) or presence of PMA (PMA) and PKC isoforms inhibitors. The levels of total and phospo-paxillin were determined by immunoblotting (A) and quantified using densitometric analysis (B). The data are expressed as fold change relative to control after normalization to loading control. PMA treatment increased paxillin phosphorylation, which was attenuated by the inhibition of PKCβII (PMA+βII), but not PKCβI (PMA+βI). (C) Cell adhesion assay was used to examine IEC-matrix adhesion after PMA treatment in the presence or absence of specific inhibitors of PKC isoforms. PMA treatment significantly increased cell-matrix adhesion.This effect was reversed with inhibition of PKCβII, but not PKCβI. *significantly different (p<0.05). (D) To examine formation of focal adhesions by the cells at the leading edge (upper panels), and cells away from the wound (bottom panels) IECs were fixed and stained for F-actin (red) and phospho-paxillin (green). Representative images show increased phosphorylation of paxillin both at the lamellipodium and areas remote from the wound after PKC activation with PMA. The bar is 20 µm. (E–F) Quantification of the changes in the relative fluorescence intensity, as an indication of the changes in paxillin phosphorylation. (E) Average relative fluorescence intensity of 5 randomly placed lines (as shown in (D), bottom panels) per field of view, at least 7 fields per condition. N = 3 independent experiments. **significantly different (p<0.01). (F) Fluorescence intensity profiles of phospho-paxillin along representative white lines projected across fields of view (as shown on the images in (D), bottom panels) that were used for quantification in (E).
Mentions: Cellular protrusions at the leading edge have to adhere to the matrix to mediate forward cell movement. Such matrix adhesions in migrating cells are referred to as focal complexes. Paxillin, is a key protein that regulates focal cell matrix adhesion dynamics [9]. Therefore we asked whether PMA stimulation of IECs results in increased activation of paxillin (increased phosphorylation, Y118), and if this event is mediated by PKCβII. Using western blot analysis we found increased phospho-paxillin (∼2.6-fod) 4 hours after PMA stimulation (Fig. 6A,B). A small non-significant increase in total paxillin level (∼1.1-fold) was observed. Inhibition of PKCβII, but not PKCβI prevented the PMA induced increase in paxillin phosphorylation consistent with the effect of PMA and PKCβII inhibition on lamellipodia formation and wound closure. Similar results were observed with another key protein that is activated in focal complexes during cell adhesion and migration, focal adhesion kinse (FAK) [46]. PMA treatment significantly increased FAK phosphorylation (Y397), which was reversed with inhibition of PKCβII, but not PKCβI (Figure S4). As both phospho-paxillin and phospho-FAK were previously reported in engaged cell-matrix adhesions [46], we next investigated the role for PMA and PKCβII in IEC adhesion to extracellular matrix. PMA treatment increased cell-matrix adhesion (∼1.8-fold). This increase was reversed in the presence of PKCβII, but not PKCβI pharmacologic inhibitors (Fig. 6C), confirming that PKCβII activation by PMA enhances assembly of focal adhesions. Furthermore, as shown in representative immunofluorescence images (Fig. 6D upper panels) PMA induced lamellipodia had increased phospho-paxillin (Y118) that was observed in aggregates suggestive of focal adhesion plaques at the leading edge of spreading IECs. Interestingly, PMA treatment also significantly increased phosphorylation of paxillin in IECs located greater than 100 µm away from the wound (Fig. 6D bottom panels, and quantification Fig. 7E,F). Epithelial cells migrate as a sheet, where cells removed from the leadings edge also contribute to wound closure by cell spreading. As paxillin and FAK play an important role in cell spreading, our findings suggest that PMA-induced activation of paxillin in IECs is accompanied by spreading of the entire epithelial sheet and not just cells at the leading edge.

Bottom Line: We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours.Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin.These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.

View Article: PubMed Central - PubMed

Affiliation: Epithelial Pathobiology and Mucosal Inflammation Research Unit, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA. ronen.sumagin@emory.edu

ABSTRACT
Rapid repair of epithelial wounds is essential for intestinal homeostasis, and involves cell proliferation and migration, which in turn are mediated by multiple cellular signaling events including PKC activation. PKC isoforms have been implicated in regulating cell proliferation and migration, however, the role of PKCs in intestinal epithelial cell (IEC) wound healing is still not completely understood. In the current work we used phorbol 12-myristate 13-acetate (PMA), a well recognized agonist of classical and non-conventional PKC subfamilies to investigate the effect of PKC activation on IEC wound healing. We found that PMA treatment of wounded IEC monolayers resulted in 5.8±0.7-fold increase in wound closure after 24 hours. The PMA effect was specifically mediated by PKCβII, as its inhibition significantly diminished the PMA-induced increase in wound closure. Furthermore, we show that the PKCβII-mediated increase in IEC wound closure after PMA stimulation was mediated by increased cell spreading/cell migration but not proliferation. Cell migration was mediated by PKCβII dependent actin cytoskeleton reorganization, enhanced formation of lamellipodial extrusions at the leading edge and increased activation of the focal adhesion protein, paxillin. These findings support a role for PKCβII in IEC wound repair and further demonstrate the ability of epithelial cells to migrate as a sheet thereby efficiently covering denuded surfaces to recover the intestinal epithelial barrier.

Show MeSH
Related in: MedlinePlus