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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 mediates extension of cell membrane protrusions in response to PMA stimulation.Scratch wounded cell monolayers were allowed to migrate for 4 hours in the absence (control) pr presence of PMA (PMA). (A) Representative images demonstrating the orientation of the Golgi apparatus in migrating cells at the wound edge with or without PMA activation. IECs were then fixed and stained for Golgi (red) and nuclei (blue). The bar is 20 µm. (B) Cell orientation in at least 8 randomly selected fields of N = 4 independent experiments were quantified as described in methods. No significant difference was observed in unstimulated versus PMA activated wounded monolayers. White arrows show correctly oriented migrated IECs. The bar is 20 µm. (C) IECs were fixed and stained for F-actin (green) and nuclei (blue). Representative images depict extended lamellipodium formation after PMA treatment, which was prevented by inhibition of PKCβII. (D) The area of the lamellipodium was defined as area past the nuclei of leading edge cells, as indicated by the white dashed line and quantified following the indicated treatment. At least 5 random fields per each condition were analyzed. N = 4 independent experiments. **significantly different (p<0.01).
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pone-0055775-g005: PKCβII mediates extension of cell membrane protrusions in response to PMA stimulation.Scratch wounded cell monolayers were allowed to migrate for 4 hours in the absence (control) pr presence of PMA (PMA). (A) Representative images demonstrating the orientation of the Golgi apparatus in migrating cells at the wound edge with or without PMA activation. IECs were then fixed and stained for Golgi (red) and nuclei (blue). The bar is 20 µm. (B) Cell orientation in at least 8 randomly selected fields of N = 4 independent experiments were quantified as described in methods. No significant difference was observed in unstimulated versus PMA activated wounded monolayers. White arrows show correctly oriented migrated IECs. The bar is 20 µm. (C) IECs were fixed and stained for F-actin (green) and nuclei (blue). Representative images depict extended lamellipodium formation after PMA treatment, which was prevented by inhibition of PKCβII. (D) The area of the lamellipodium was defined as area past the nuclei of leading edge cells, as indicated by the white dashed line and quantified following the indicated treatment. At least 5 random fields per each condition were analyzed. N = 4 independent experiments. **significantly different (p<0.01).

Mentions: Polarization of the leading edge, orientation of the Golgi apparatus in the direction of migration, and lamellipodial extrusions at the leading edge are required for forward epithelial cell movement [7]. To determine IEC polarization in the direction of migration, we first examined the localization of Golgi apparatus in control and PMA activated migrating T84 IECs. We did not observe significant differences in the directional polarity of the leading edge in PMA stimulated cells vs. control migrating IECs (Fig. 5A,B). Next we examined lamellipodium formation in control and PMA activated migrating T84 IECs. Prominent lamellipodia were observed as early as 4 hours after wounding in both control and PMA activated migrating cells. However, PKC activation by PMA resulted in significantly larger lamellipodia at the leading edge compared to those observed in control migrating cells (area between the nuclei and the cell edge as indicated by the white dashed line, 1565±56.8 vs 1197.2±65.6 µm2, respectively, Fig. 5D, and representative images Fig. 5C). Inhibition of PKCβII but not PKCβI significantly attenuated lamellipodia formation, supporting a role of these PKC isoforms in mediating cellular protrusions at 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 extension of cell membrane protrusions in response to PMA stimulation.Scratch wounded cell monolayers were allowed to migrate for 4 hours in the absence (control) pr presence of PMA (PMA). (A) Representative images demonstrating the orientation of the Golgi apparatus in migrating cells at the wound edge with or without PMA activation. IECs were then fixed and stained for Golgi (red) and nuclei (blue). The bar is 20 µm. (B) Cell orientation in at least 8 randomly selected fields of N = 4 independent experiments were quantified as described in methods. No significant difference was observed in unstimulated versus PMA activated wounded monolayers. White arrows show correctly oriented migrated IECs. The bar is 20 µm. (C) IECs were fixed and stained for F-actin (green) and nuclei (blue). Representative images depict extended lamellipodium formation after PMA treatment, which was prevented by inhibition of PKCβII. (D) The area of the lamellipodium was defined as area past the nuclei of leading edge cells, as indicated by the white dashed line and quantified following the indicated treatment. At least 5 random fields per each condition were analyzed. N = 4 independent experiments. **significantly different (p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569445&req=5

pone-0055775-g005: PKCβII mediates extension of cell membrane protrusions in response to PMA stimulation.Scratch wounded cell monolayers were allowed to migrate for 4 hours in the absence (control) pr presence of PMA (PMA). (A) Representative images demonstrating the orientation of the Golgi apparatus in migrating cells at the wound edge with or without PMA activation. IECs were then fixed and stained for Golgi (red) and nuclei (blue). The bar is 20 µm. (B) Cell orientation in at least 8 randomly selected fields of N = 4 independent experiments were quantified as described in methods. No significant difference was observed in unstimulated versus PMA activated wounded monolayers. White arrows show correctly oriented migrated IECs. The bar is 20 µm. (C) IECs were fixed and stained for F-actin (green) and nuclei (blue). Representative images depict extended lamellipodium formation after PMA treatment, which was prevented by inhibition of PKCβII. (D) The area of the lamellipodium was defined as area past the nuclei of leading edge cells, as indicated by the white dashed line and quantified following the indicated treatment. At least 5 random fields per each condition were analyzed. N = 4 independent experiments. **significantly different (p<0.01).
Mentions: Polarization of the leading edge, orientation of the Golgi apparatus in the direction of migration, and lamellipodial extrusions at the leading edge are required for forward epithelial cell movement [7]. To determine IEC polarization in the direction of migration, we first examined the localization of Golgi apparatus in control and PMA activated migrating T84 IECs. We did not observe significant differences in the directional polarity of the leading edge in PMA stimulated cells vs. control migrating IECs (Fig. 5A,B). Next we examined lamellipodium formation in control and PMA activated migrating T84 IECs. Prominent lamellipodia were observed as early as 4 hours after wounding in both control and PMA activated migrating cells. However, PKC activation by PMA resulted in significantly larger lamellipodia at the leading edge compared to those observed in control migrating cells (area between the nuclei and the cell edge as indicated by the white dashed line, 1565±56.8 vs 1197.2±65.6 µm2, respectively, Fig. 5D, and representative images Fig. 5C). Inhibition of PKCβII but not PKCβI significantly attenuated lamellipodia formation, supporting a role of these PKC isoforms in mediating cellular protrusions at 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