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

PKC activation with PMA enhances epithelial wound healing.(A) Confluent T84 IEC monolayers were wounded by introduction of a single linear scratch wound. Wound closure was measured over 48 h in unstimulated (control) and PMA activated IEC monolayers as detailed in the methods section. (B) Representative images of unstimulated (control, left panels) and PMA activated (PMA, 200 nM right panels) IEC monolayers immediately after wounding (0 h, upper panels) and after 48 hours (48 h, bottom panels). PMA treatment induced a dramatic increase in wound closure as early as 12 and 24 hours. The bar is 50 µm. N = 4 independent experiments. **significantly different from each other (p<0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569445&req=5

pone-0055775-g001: PKC activation with PMA enhances epithelial wound healing.(A) Confluent T84 IEC monolayers were wounded by introduction of a single linear scratch wound. Wound closure was measured over 48 h in unstimulated (control) and PMA activated IEC monolayers as detailed in the methods section. (B) Representative images of unstimulated (control, left panels) and PMA activated (PMA, 200 nM right panels) IEC monolayers immediately after wounding (0 h, upper panels) and after 48 hours (48 h, bottom panels). PMA treatment induced a dramatic increase in wound closure as early as 12 and 24 hours. The bar is 50 µm. N = 4 independent experiments. **significantly different from each other (p<0.01).

Mentions: PMA activation of PKC isoforms has been shown to enhance epithelial cell growth, proliferation, as well as motility of myeloid cells and fibroblasts [28], [38]. Since epithelial wound healing requires cell migration and proliferation, we hypothesized that PKC activation promotes IEC wound closure. To test this possibility, confluent T84 cells were mechanically wounded (see Methods section) and healing was assessed in the presence or absence of PKC activator, PMA. We observed that in the presence of PMA (200 nM) T84 cell wound healing was dramatically enhanced, 15.9±2.6 fold after 12 hours and 5.8±0.7 after 24 hours (Fig. 1A). Consequently, 24 hours after wounding PMA treated monolayers have recovered 88.0±2.5% of the wounded area compared to 16.2±1.6% in non-treated monolayers. After 48 hours all wounds in PMA treated monolayers were healed, however only 48.2±4.5% of the wounded area recovered in non-treated monolayers (Fig. 1A, and representative images, Fig. 1B). The effect of PMA on wound healing was also confirmed in a different intestinal epithelial cell line, Caco2 (Figure S1). These findings clearly support a role of PKCs in epithelial wound healing.


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)

PKC activation with PMA enhances epithelial wound healing.(A) Confluent T84 IEC monolayers were wounded by introduction of a single linear scratch wound. Wound closure was measured over 48 h in unstimulated (control) and PMA activated IEC monolayers as detailed in the methods section. (B) Representative images of unstimulated (control, left panels) and PMA activated (PMA, 200 nM right panels) IEC monolayers immediately after wounding (0 h, upper panels) and after 48 hours (48 h, bottom panels). PMA treatment induced a dramatic increase in wound closure as early as 12 and 24 hours. The bar is 50 µm. N = 4 independent experiments. **significantly different from each other (p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055775-g001: PKC activation with PMA enhances epithelial wound healing.(A) Confluent T84 IEC monolayers were wounded by introduction of a single linear scratch wound. Wound closure was measured over 48 h in unstimulated (control) and PMA activated IEC monolayers as detailed in the methods section. (B) Representative images of unstimulated (control, left panels) and PMA activated (PMA, 200 nM right panels) IEC monolayers immediately after wounding (0 h, upper panels) and after 48 hours (48 h, bottom panels). PMA treatment induced a dramatic increase in wound closure as early as 12 and 24 hours. The bar is 50 µm. N = 4 independent experiments. **significantly different from each other (p<0.01).
Mentions: PMA activation of PKC isoforms has been shown to enhance epithelial cell growth, proliferation, as well as motility of myeloid cells and fibroblasts [28], [38]. Since epithelial wound healing requires cell migration and proliferation, we hypothesized that PKC activation promotes IEC wound closure. To test this possibility, confluent T84 cells were mechanically wounded (see Methods section) and healing was assessed in the presence or absence of PKC activator, PMA. We observed that in the presence of PMA (200 nM) T84 cell wound healing was dramatically enhanced, 15.9±2.6 fold after 12 hours and 5.8±0.7 after 24 hours (Fig. 1A). Consequently, 24 hours after wounding PMA treated monolayers have recovered 88.0±2.5% of the wounded area compared to 16.2±1.6% in non-treated monolayers. After 48 hours all wounds in PMA treated monolayers were healed, however only 48.2±4.5% of the wounded area recovered in non-treated monolayers (Fig. 1A, and representative images, Fig. 1B). The effect of PMA on wound healing was also confirmed in a different intestinal epithelial cell line, Caco2 (Figure S1). These findings clearly support a role of PKCs in epithelial wound healing.

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