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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 translocates to the cell membrane upon PMA treatment.Cellular localization of selected classical PKCs was determined by immnoblotting (A) and densitometric analysis (B) of the total (T), the cytosolic (C), and the membrane (M) fractions in unstimulated (control) and PMA activated (PMA, 200 nM, 4 h) IECs. **significantly different (p<0.01). N = 4 independent experiments. Following PMA treatment PKCβII translocated to the cell membrane, indicative of its activation. (C) PKC isoform localization was also confirmed by immunofluorescence labeling and confocal microscopy. The images show translocation of PKCβII from the cytosol (control) to cell membrane (PMA treatment), and are representative of 3 independent experiments. The bar is 20 µm.
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pone-0055775-g003: PKCβII translocates to the cell membrane upon PMA treatment.Cellular localization of selected classical PKCs was determined by immnoblotting (A) and densitometric analysis (B) of the total (T), the cytosolic (C), and the membrane (M) fractions in unstimulated (control) and PMA activated (PMA, 200 nM, 4 h) IECs. **significantly different (p<0.01). N = 4 independent experiments. Following PMA treatment PKCβII translocated to the cell membrane, indicative of its activation. (C) PKC isoform localization was also confirmed by immunofluorescence labeling and confocal microscopy. The images show translocation of PKCβII from the cytosol (control) to cell membrane (PMA treatment), and are representative of 3 independent experiments. The bar is 20 µm.

Mentions: A hallmark of PKC activation is its translocation to the membrane where it interacts with DAG and becomes functionally active [15]. Since we determined that PMA enhanced wound closure was primarily mediated by PKCβII, we next examined the cellular localization of PKCβII, as well as other classical PKCs, PKCβI and PKCγ in T84 IECs stimulated with PMA for 4 hours. As evident from western blot analysis of cytosolic and membrane fractions of T84 IECs (Fig. 3A,B), and confirmed by immunofluorescence labeling and confocal microscopy (Fig. 3C) PKCβII under basal conditions was found to mostly reside in the cytosol, however upon PMA treatment PKCβII translocation to cell membrane was observed (18.1±4.2% at the membrane, control vs. 45.1±2.4% at the membrane, PMA, Fig. 3B), indicative of its activation. PKCβI was found both in the cytosolic and to a lesser extent the membrane fractions and its distribution was not changed after PMA treatment. Similarly, the localization of PKCγ in the cytosolic fraction under basal conditions was not changed with PMA treatment (Fig. 3). Furthermore, we confirmed that PKCβII translocation to cell membrane occurs in leading edge cells after PMA treatment, but not in non-treated wounds (Figure S3C).


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 translocates to the cell membrane upon PMA treatment.Cellular localization of selected classical PKCs was determined by immnoblotting (A) and densitometric analysis (B) of the total (T), the cytosolic (C), and the membrane (M) fractions in unstimulated (control) and PMA activated (PMA, 200 nM, 4 h) IECs. **significantly different (p<0.01). N = 4 independent experiments. Following PMA treatment PKCβII translocated to the cell membrane, indicative of its activation. (C) PKC isoform localization was also confirmed by immunofluorescence labeling and confocal microscopy. The images show translocation of PKCβII from the cytosol (control) to cell membrane (PMA treatment), and are representative of 3 independent experiments. The bar is 20 µm.
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Related In: Results  -  Collection

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pone-0055775-g003: PKCβII translocates to the cell membrane upon PMA treatment.Cellular localization of selected classical PKCs was determined by immnoblotting (A) and densitometric analysis (B) of the total (T), the cytosolic (C), and the membrane (M) fractions in unstimulated (control) and PMA activated (PMA, 200 nM, 4 h) IECs. **significantly different (p<0.01). N = 4 independent experiments. Following PMA treatment PKCβII translocated to the cell membrane, indicative of its activation. (C) PKC isoform localization was also confirmed by immunofluorescence labeling and confocal microscopy. The images show translocation of PKCβII from the cytosol (control) to cell membrane (PMA treatment), and are representative of 3 independent experiments. The bar is 20 µm.
Mentions: A hallmark of PKC activation is its translocation to the membrane where it interacts with DAG and becomes functionally active [15]. Since we determined that PMA enhanced wound closure was primarily mediated by PKCβII, we next examined the cellular localization of PKCβII, as well as other classical PKCs, PKCβI and PKCγ in T84 IECs stimulated with PMA for 4 hours. As evident from western blot analysis of cytosolic and membrane fractions of T84 IECs (Fig. 3A,B), and confirmed by immunofluorescence labeling and confocal microscopy (Fig. 3C) PKCβII under basal conditions was found to mostly reside in the cytosol, however upon PMA treatment PKCβII translocation to cell membrane was observed (18.1±4.2% at the membrane, control vs. 45.1±2.4% at the membrane, PMA, Fig. 3B), indicative of its activation. PKCβI was found both in the cytosolic and to a lesser extent the membrane fractions and its distribution was not changed after PMA treatment. Similarly, the localization of PKCγ in the cytosolic fraction under basal conditions was not changed with PMA treatment (Fig. 3). Furthermore, we confirmed that PKCβII translocation to cell membrane occurs in leading edge cells after PMA treatment, but not in non-treated wounds (Figure S3C).

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