Limits...
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.

Show MeSH

Related in: MedlinePlus

PKCβII mediates PMA induced epithelial cell spreading.(A,B) Subconfluent IECs monolayers (∼40% confluency) were allowed to spread on 13-mm collagen-coated coverslips in the absence (left panels) or presence (right panels) of PMA activation (200nM, 4 h), with the addition of PKCβII or PKCβI inhibitors as indicated. Cell area per field of view was measured before (t = 0 h, upper panels) and after treatment (t = 4 h, bottom panels). (A) Representative images demonstrating enhanced IEC spreading after PMA treatment. The bar is 50 µm. (B) Quantification of IEC spreading in the presence or absence of PKCβII (PMA+βII) or PKCβI (PMA+βI) inhibitors. Data presented as percent cell area/total area of the field of view. PMA induced cell spreading was significantly attenuated after inhibition of PKCβII. **significantly different (p<0.01). (C) Spreading IECs stimulated with PMA were fixed and stained for F-actin at t = 0 (left panels) and t = 4 h (right panels). Representative confocal microscopy images show most apical (upper panels) and most basal (bottom panels) distribution of F-actin. A dramatic reorganization of F-actin including loss of perijunctional actomyosin belt at the apical side, and redistribution of actin towards cell borders, as well as loss of stress fibers at the cell basal side in response to PMA treatment was observed. The bar is 20 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569445&req=5

pone-0055775-g007: PKCβII mediates PMA induced epithelial cell spreading.(A,B) Subconfluent IECs monolayers (∼40% confluency) were allowed to spread on 13-mm collagen-coated coverslips in the absence (left panels) or presence (right panels) of PMA activation (200nM, 4 h), with the addition of PKCβII or PKCβI inhibitors as indicated. Cell area per field of view was measured before (t = 0 h, upper panels) and after treatment (t = 4 h, bottom panels). (A) Representative images demonstrating enhanced IEC spreading after PMA treatment. The bar is 50 µm. (B) Quantification of IEC spreading in the presence or absence of PKCβII (PMA+βII) or PKCβI (PMA+βI) inhibitors. Data presented as percent cell area/total area of the field of view. PMA induced cell spreading was significantly attenuated after inhibition of PKCβII. **significantly different (p<0.01). (C) Spreading IECs stimulated with PMA were fixed and stained for F-actin at t = 0 (left panels) and t = 4 h (right panels). Representative confocal microscopy images show most apical (upper panels) and most basal (bottom panels) distribution of F-actin. A dramatic reorganization of F-actin including loss of perijunctional actomyosin belt at the apical side, and redistribution of actin towards cell borders, as well as loss of stress fibers at the cell basal side in response to PMA treatment was observed. The bar is 20 µm.

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 mediates PMA induced epithelial cell spreading.(A,B) Subconfluent IECs monolayers (∼40% confluency) were allowed to spread on 13-mm collagen-coated coverslips in the absence (left panels) or presence (right panels) of PMA activation (200nM, 4 h), with the addition of PKCβII or PKCβI inhibitors as indicated. Cell area per field of view was measured before (t = 0 h, upper panels) and after treatment (t = 4 h, bottom panels). (A) Representative images demonstrating enhanced IEC spreading after PMA treatment. The bar is 50 µm. (B) Quantification of IEC spreading in the presence or absence of PKCβII (PMA+βII) or PKCβI (PMA+βI) inhibitors. Data presented as percent cell area/total area of the field of view. PMA induced cell spreading was significantly attenuated after inhibition of PKCβII. **significantly different (p<0.01). (C) Spreading IECs stimulated with PMA were fixed and stained for F-actin at t = 0 (left panels) and t = 4 h (right panels). Representative confocal microscopy images show most apical (upper panels) and most basal (bottom panels) distribution of F-actin. A dramatic reorganization of F-actin including loss of perijunctional actomyosin belt at the apical side, and redistribution of actin towards cell borders, as well as loss of stress fibers at the cell basal side in response to PMA treatment was observed. The bar is 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569445&req=5

pone-0055775-g007: PKCβII mediates PMA induced epithelial cell spreading.(A,B) Subconfluent IECs monolayers (∼40% confluency) were allowed to spread on 13-mm collagen-coated coverslips in the absence (left panels) or presence (right panels) of PMA activation (200nM, 4 h), with the addition of PKCβII or PKCβI inhibitors as indicated. Cell area per field of view was measured before (t = 0 h, upper panels) and after treatment (t = 4 h, bottom panels). (A) Representative images demonstrating enhanced IEC spreading after PMA treatment. The bar is 50 µm. (B) Quantification of IEC spreading in the presence or absence of PKCβII (PMA+βII) or PKCβI (PMA+βI) inhibitors. Data presented as percent cell area/total area of the field of view. PMA induced cell spreading was significantly attenuated after inhibition of PKCβII. **significantly different (p<0.01). (C) Spreading IECs stimulated with PMA were fixed and stained for F-actin at t = 0 (left panels) and t = 4 h (right panels). Representative confocal microscopy images show most apical (upper panels) and most basal (bottom panels) distribution of F-actin. A dramatic reorganization of F-actin including loss of perijunctional actomyosin belt at the apical side, and redistribution of actin towards cell borders, as well as loss of stress fibers at the cell basal side in response to PMA treatment was observed. The bar is 20 µm.
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