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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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Enhanced T84 cell wound closure following PMA activation was not due to increased cell proliferation.Cell proliferation in non-wounded versus wounded IEC monlayers, unstimulated or after PMA activation (200 nM, 4 h) was assayed using cell cycle analysis (A,B) and Edu incorporation (D,E). The distribution of cells in G1 and G2/M phases under indicated conditions is demonstrated by the representative flow diagrams (A) and summarized in (B). PMA treatment significantly increased the number of cells in G2/M phase in non-wounded IEC monolayers, however, there was no significant effect on proliferation of migrating IECs. *significantly different from control (p<0.05). n.s, not significant. N = 4 independent experiments. (C) In separate experiments wounded cell monolayers were pretreated with L-mimosine (100 µM, 12 h) stimulated with PMA, and analyzed for cell cycle distribution as described above and compared to unstimulated (control) cells. L-mimosine treatment significantly diminished cell proliferation in scratch wounded cell monolayers, both with and without PMA activation. **significantly different from no treatment (p<0.01). (D–E) Cell proliferation at the wound edge was assessed by Edu incorporation. Scratch wounded or confluent cell monolayers were treated with PMA for 4 hr at 37°C before addition of EdU. At least 5 random fields per each condition were analyzed; the data are presented as % proliferating (nuclei stained in purple, EdU)/of total cells (nuclei stained in blue, topro-3) in the field. As shown in representative images (D) and quantified in (E) PKC activation with PMA had no effect on cell proliferation at the edge of the wound. N = 3 independent experiments. The bar is 50 µm. (F) Scratch wounded cell monolayers were either pretreated with L-mimosine (100 µM, 12 h) prior to PMA activation or activated with PMA alone, and wound closure was quantified after 24 hours and compared to unstimulated (control) cells. While L-mimosine treatment significantly decreased wound closure, it accounted only for a small portion of the PMA effect. **significantly different from each other (p<0.01).
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pone-0055775-g004: Enhanced T84 cell wound closure following PMA activation was not due to increased cell proliferation.Cell proliferation in non-wounded versus wounded IEC monlayers, unstimulated or after PMA activation (200 nM, 4 h) was assayed using cell cycle analysis (A,B) and Edu incorporation (D,E). The distribution of cells in G1 and G2/M phases under indicated conditions is demonstrated by the representative flow diagrams (A) and summarized in (B). PMA treatment significantly increased the number of cells in G2/M phase in non-wounded IEC monolayers, however, there was no significant effect on proliferation of migrating IECs. *significantly different from control (p<0.05). n.s, not significant. N = 4 independent experiments. (C) In separate experiments wounded cell monolayers were pretreated with L-mimosine (100 µM, 12 h) stimulated with PMA, and analyzed for cell cycle distribution as described above and compared to unstimulated (control) cells. L-mimosine treatment significantly diminished cell proliferation in scratch wounded cell monolayers, both with and without PMA activation. **significantly different from no treatment (p<0.01). (D–E) Cell proliferation at the wound edge was assessed by Edu incorporation. Scratch wounded or confluent cell monolayers were treated with PMA for 4 hr at 37°C before addition of EdU. At least 5 random fields per each condition were analyzed; the data are presented as % proliferating (nuclei stained in purple, EdU)/of total cells (nuclei stained in blue, topro-3) in the field. As shown in representative images (D) and quantified in (E) PKC activation with PMA had no effect on cell proliferation at the edge of the wound. N = 3 independent experiments. The bar is 50 µm. (F) Scratch wounded cell monolayers were either pretreated with L-mimosine (100 µM, 12 h) prior to PMA activation or activated with PMA alone, and wound closure was quantified after 24 hours and compared to unstimulated (control) cells. While L-mimosine treatment significantly decreased wound closure, it accounted only for a small portion of the PMA effect. **significantly different from each other (p<0.01).

Mentions: Since cell proliferation contributes to IEC wound repair, and PMA has been shown to enhance cell proliferation, we next examined whether PMA enhanced IEC wound healing was due to increased IEC proliferation. To assess the effect of PMA on cell proliferation we used Propidium Iodide staining and flow cytometry of scratch wounded T84 monolayers in the presence or absence of PMA. Consistent with the finding in other cultured epithelial cells [44], PMA treatment of confluent T84 monolayers (non-migrating IECs) significantly increased the number of cells in G2/M phase (from 26.2±1.4%, control to 37.1±2.4%, PMA, Fig. 4A,B) indicative of increased cell proliferation. Interestingly, wounding itself enhanced cell proliferation (34.6±2.1%, Fig. 4A,B), analogous to that observed with PMA treatment. Importantly, PMA stimulation of wounded monolayers failed to further potentiate the increase in cell proliferation, suggesting that PMA has no further effect on proliferation of migrating IECs. These findings were also confirmed by quantifying Edu incorporation into actively proliferating epithelial cells. The number of proliferating cells at the sites of closing wounds (wound edge) was not significantly different between untreated and PMA stimulated wounded monolayers (Fig. 4E and representative images, Fig. 4D). Together these findings suggest that while PKCs might play a role in epithelial cell growth and survival, PMA effects on wound healing was not due to increased cell proliferation. To further confirm that IEC proliferation does not play a major role in PMA-induced increase in wound closure we used L-mimosine, which, has been previously shown to inhibit cells at a regulatory step in late G1 phase, thus preventing cell proliferation [45]. Indeed inhibition of cell proliferation using L-mimosine (L-mim) accounted only for a small fraction (∼20%) of the ∼70% total increase in wound closure after 24 hours PMA activation (Fig. 4F). L-mimosine treatment significantly diminished cell proliferation in resealing wounds, both with and without PMA activation (Fig. 4C) confirming an effect of this inhibitor on cell proliferation.


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)

Enhanced T84 cell wound closure following PMA activation was not due to increased cell proliferation.Cell proliferation in non-wounded versus wounded IEC monlayers, unstimulated or after PMA activation (200 nM, 4 h) was assayed using cell cycle analysis (A,B) and Edu incorporation (D,E). The distribution of cells in G1 and G2/M phases under indicated conditions is demonstrated by the representative flow diagrams (A) and summarized in (B). PMA treatment significantly increased the number of cells in G2/M phase in non-wounded IEC monolayers, however, there was no significant effect on proliferation of migrating IECs. *significantly different from control (p<0.05). n.s, not significant. N = 4 independent experiments. (C) In separate experiments wounded cell monolayers were pretreated with L-mimosine (100 µM, 12 h) stimulated with PMA, and analyzed for cell cycle distribution as described above and compared to unstimulated (control) cells. L-mimosine treatment significantly diminished cell proliferation in scratch wounded cell monolayers, both with and without PMA activation. **significantly different from no treatment (p<0.01). (D–E) Cell proliferation at the wound edge was assessed by Edu incorporation. Scratch wounded or confluent cell monolayers were treated with PMA for 4 hr at 37°C before addition of EdU. At least 5 random fields per each condition were analyzed; the data are presented as % proliferating (nuclei stained in purple, EdU)/of total cells (nuclei stained in blue, topro-3) in the field. As shown in representative images (D) and quantified in (E) PKC activation with PMA had no effect on cell proliferation at the edge of the wound. N = 3 independent experiments. The bar is 50 µm. (F) Scratch wounded cell monolayers were either pretreated with L-mimosine (100 µM, 12 h) prior to PMA activation or activated with PMA alone, and wound closure was quantified after 24 hours and compared to unstimulated (control) cells. While L-mimosine treatment significantly decreased wound closure, it accounted only for a small portion of the PMA effect. **significantly different from each other (p<0.01).
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Related In: Results  -  Collection

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pone-0055775-g004: Enhanced T84 cell wound closure following PMA activation was not due to increased cell proliferation.Cell proliferation in non-wounded versus wounded IEC monlayers, unstimulated or after PMA activation (200 nM, 4 h) was assayed using cell cycle analysis (A,B) and Edu incorporation (D,E). The distribution of cells in G1 and G2/M phases under indicated conditions is demonstrated by the representative flow diagrams (A) and summarized in (B). PMA treatment significantly increased the number of cells in G2/M phase in non-wounded IEC monolayers, however, there was no significant effect on proliferation of migrating IECs. *significantly different from control (p<0.05). n.s, not significant. N = 4 independent experiments. (C) In separate experiments wounded cell monolayers were pretreated with L-mimosine (100 µM, 12 h) stimulated with PMA, and analyzed for cell cycle distribution as described above and compared to unstimulated (control) cells. L-mimosine treatment significantly diminished cell proliferation in scratch wounded cell monolayers, both with and without PMA activation. **significantly different from no treatment (p<0.01). (D–E) Cell proliferation at the wound edge was assessed by Edu incorporation. Scratch wounded or confluent cell monolayers were treated with PMA for 4 hr at 37°C before addition of EdU. At least 5 random fields per each condition were analyzed; the data are presented as % proliferating (nuclei stained in purple, EdU)/of total cells (nuclei stained in blue, topro-3) in the field. As shown in representative images (D) and quantified in (E) PKC activation with PMA had no effect on cell proliferation at the edge of the wound. N = 3 independent experiments. The bar is 50 µm. (F) Scratch wounded cell monolayers were either pretreated with L-mimosine (100 µM, 12 h) prior to PMA activation or activated with PMA alone, and wound closure was quantified after 24 hours and compared to unstimulated (control) cells. While L-mimosine treatment significantly decreased wound closure, it accounted only for a small portion of the PMA effect. **significantly different from each other (p<0.01).
Mentions: Since cell proliferation contributes to IEC wound repair, and PMA has been shown to enhance cell proliferation, we next examined whether PMA enhanced IEC wound healing was due to increased IEC proliferation. To assess the effect of PMA on cell proliferation we used Propidium Iodide staining and flow cytometry of scratch wounded T84 monolayers in the presence or absence of PMA. Consistent with the finding in other cultured epithelial cells [44], PMA treatment of confluent T84 monolayers (non-migrating IECs) significantly increased the number of cells in G2/M phase (from 26.2±1.4%, control to 37.1±2.4%, PMA, Fig. 4A,B) indicative of increased cell proliferation. Interestingly, wounding itself enhanced cell proliferation (34.6±2.1%, Fig. 4A,B), analogous to that observed with PMA treatment. Importantly, PMA stimulation of wounded monolayers failed to further potentiate the increase in cell proliferation, suggesting that PMA has no further effect on proliferation of migrating IECs. These findings were also confirmed by quantifying Edu incorporation into actively proliferating epithelial cells. The number of proliferating cells at the sites of closing wounds (wound edge) was not significantly different between untreated and PMA stimulated wounded monolayers (Fig. 4E and representative images, Fig. 4D). Together these findings suggest that while PKCs might play a role in epithelial cell growth and survival, PMA effects on wound healing was not due to increased cell proliferation. To further confirm that IEC proliferation does not play a major role in PMA-induced increase in wound closure we used L-mimosine, which, has been previously shown to inhibit cells at a regulatory step in late G1 phase, thus preventing cell proliferation [45]. Indeed inhibition of cell proliferation using L-mimosine (L-mim) accounted only for a small fraction (∼20%) of the ∼70% total increase in wound closure after 24 hours PMA activation (Fig. 4F). L-mimosine treatment significantly diminished cell proliferation in resealing wounds, both with and without PMA activation (Fig. 4C) confirming an effect of this inhibitor on cell proliferation.

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