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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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Spreading IECs were fixed and stained for E-Cadherin before (control) and after PMA activation (PMA) in the presence or absence of PKC blockers as indicated.(A) Representative confocal images acquired in series in Z-direction show changes in cell height before (control, 1–7) and after PMA treatment (PMA, 1–3). The bar is 20 µm. (B) Quantification of cell height in 7 randomly selected fields per condition, N = 3 independent experiments. **significantly different (p<0.01). (C) The changes in the surface area of individual cells under the specified conditions were quantified from images obtained using confocal microscopy, and positioning the focal plane at the apical IEC surface, and depicted in the representative images (D). The bar is 10 µm. The height of IECs was significantly decreased, and the approximate cell surface area was significantly increased after PKC activation by PMA consistent with cell spreading. In both cases inhibition of PKCβII significantly attenuated these changes. **significantly different (p<0.01).
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pone-0055775-g008: Spreading IECs were fixed and stained for E-Cadherin before (control) and after PMA activation (PMA) in the presence or absence of PKC blockers as indicated.(A) Representative confocal images acquired in series in Z-direction show changes in cell height before (control, 1–7) and after PMA treatment (PMA, 1–3). The bar is 20 µm. (B) Quantification of cell height in 7 randomly selected fields per condition, N = 3 independent experiments. **significantly different (p<0.01). (C) The changes in the surface area of individual cells under the specified conditions were quantified from images obtained using confocal microscopy, and positioning the focal plane at the apical IEC surface, and depicted in the representative images (D). The bar is 10 µm. The height of IECs was significantly decreased, and the approximate cell surface area was significantly increased after PKC activation by PMA consistent with cell spreading. In both cases inhibition of PKCβII significantly attenuated these changes. **significantly different (p<0.01).

Mentions: We further confirmed the effect of PKC activation on cell spreading by detailed examination of cell morphology in control and PMA activated subconfluent and spreading T84 cells. Confocal microscopy and serial image acquisition in X-Z plane of IECs immunostained for the adherens junction protein, E-cadherin revealed that control cell height was 7.2±0.3 µm, while cells exposed to PMA were significantly flattened (cell height, 3.2±0.3 µm, Fig. 8B and Fig. 8A, 1–7 and Fig. 8A, 1–3, respectively). Additionally, the surface area of individual spreading cells was significantly increased after PMA treatment (from 114.0±8.9 to 279.1±15.2 µm2, Fig. 8C, and representative images Fig. 8D) indicative of cell spreading. In agreement with cell spreading assay results (Fig. 7A,B), inhibition of PKCβII attenuated both the decrease in cell height and the increase in cell surface area, confirming the role for PKCβII in epithelial cell spreading.


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)

Spreading IECs were fixed and stained for E-Cadherin before (control) and after PMA activation (PMA) in the presence or absence of PKC blockers as indicated.(A) Representative confocal images acquired in series in Z-direction show changes in cell height before (control, 1–7) and after PMA treatment (PMA, 1–3). The bar is 20 µm. (B) Quantification of cell height in 7 randomly selected fields per condition, N = 3 independent experiments. **significantly different (p<0.01). (C) The changes in the surface area of individual cells under the specified conditions were quantified from images obtained using confocal microscopy, and positioning the focal plane at the apical IEC surface, and depicted in the representative images (D). The bar is 10 µm. The height of IECs was significantly decreased, and the approximate cell surface area was significantly increased after PKC activation by PMA consistent with cell spreading. In both cases inhibition of PKCβII significantly attenuated these changes. **significantly different (p<0.01).
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pone-0055775-g008: Spreading IECs were fixed and stained for E-Cadherin before (control) and after PMA activation (PMA) in the presence or absence of PKC blockers as indicated.(A) Representative confocal images acquired in series in Z-direction show changes in cell height before (control, 1–7) and after PMA treatment (PMA, 1–3). The bar is 20 µm. (B) Quantification of cell height in 7 randomly selected fields per condition, N = 3 independent experiments. **significantly different (p<0.01). (C) The changes in the surface area of individual cells under the specified conditions were quantified from images obtained using confocal microscopy, and positioning the focal plane at the apical IEC surface, and depicted in the representative images (D). The bar is 10 µm. The height of IECs was significantly decreased, and the approximate cell surface area was significantly increased after PKC activation by PMA consistent with cell spreading. In both cases inhibition of PKCβII significantly attenuated these changes. **significantly different (p<0.01).
Mentions: We further confirmed the effect of PKC activation on cell spreading by detailed examination of cell morphology in control and PMA activated subconfluent and spreading T84 cells. Confocal microscopy and serial image acquisition in X-Z plane of IECs immunostained for the adherens junction protein, E-cadherin revealed that control cell height was 7.2±0.3 µm, while cells exposed to PMA were significantly flattened (cell height, 3.2±0.3 µm, Fig. 8B and Fig. 8A, 1–7 and Fig. 8A, 1–3, respectively). Additionally, the surface area of individual spreading cells was significantly increased after PMA treatment (from 114.0±8.9 to 279.1±15.2 µm2, Fig. 8C, and representative images Fig. 8D) indicative of cell spreading. In agreement with cell spreading assay results (Fig. 7A,B), inhibition of PKCβII attenuated both the decrease in cell height and the increase in cell surface area, confirming the role for PKCβII in epithelial cell spreading.

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