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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 PMA induced increase in wound closure.Confluent IEC monolayers were wounded by introduction of a single linear scratch wound, preincubated with the specified PKC inhibitors for 1 hour (Cheleretrine, pan PKC inhibitor (5 µM, pan); Calphostin C, Classical/non-conventional PKC inhibitor (10 µM, Clas/Non-C); KIC1-1, classical PKC inhibitor (5 µM, Clas); KIBI31-1, PKCβI inhibitor (1 µM, PKCβI); KIBII31-1 (5 µM, PKCβII); KIG31-1, PKCγ inhibitor (5 µM, PKCβγ) and stimulated with PMA (200nM). The area of the wound was measured at 12 and 24 hours. Inhibition of PKCβII, but not inhibition of other members of the classical PKCs significantly diminished PMA enhanced wound closure. N = 3 independent experiments. **significantly different from control (PMA alone, p<0.01).
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pone-0055775-g002: PKCβII mediates PMA induced increase in wound closure.Confluent IEC monolayers were wounded by introduction of a single linear scratch wound, preincubated with the specified PKC inhibitors for 1 hour (Cheleretrine, pan PKC inhibitor (5 µM, pan); Calphostin C, Classical/non-conventional PKC inhibitor (10 µM, Clas/Non-C); KIC1-1, classical PKC inhibitor (5 µM, Clas); KIBI31-1, PKCβI inhibitor (1 µM, PKCβI); KIBII31-1 (5 µM, PKCβII); KIG31-1, PKCγ inhibitor (5 µM, PKCβγ) and stimulated with PMA (200nM). The area of the wound was measured at 12 and 24 hours. Inhibition of PKCβII, but not inhibition of other members of the classical PKCs significantly diminished PMA enhanced wound closure. N = 3 independent experiments. **significantly different from control (PMA alone, p<0.01).

Mentions: PMA activates both classical and non-conventional PKC subfamilies [39]. Thus to examine which PKC subfamily member promotes PMA mediated wound closure we used a panel of broad PKC inhibitors including, the pan PKC inhibitor (Cheleretrine, 5 µM [40]), the inhibitor of classical/non-conventional PKC isoforms, Calphostin C (10 µM) [41] and pan classical PKC inhibitors KIC1-1(5 µM) [36] and Go6976 (0.5 µM) [42]. Since the major effect of PMA on IECs wound closure was observed within the first 24 hours (Fig. 1A) we determined the effect of specific PKC inhibitors at 12 h and 24 h post injury. As shown in Figure 2 pan PKC inhibition as well as inhibition of classical and non-conventional PKCs reversed the PMA-induced increase in T84 IECs wound closure. Importantly, inhibition of classical PKCs alone was sufficient to inhibit the PMA-induced increase in T84 cell wound closure (Fig. 2), confirming that member/s of the classical PKC subfamily are primarily responsible for the PMA effect. Next we used PKC isoform-specific peptide inhibitors for selected classical PKCs including PKCβI (KIBI31-1, 1 µM [37]), PKCβII, (KIBII31-1, 5 µM) and PKCγ (KIG31-1, 5 µM) to identify the specific PKC isoforms that mediate the PMA effect on IEC wound closure. PKCγ and PKCβI inhibition had no significant effect on PMA induced wound closure. However, in contrast, PKCβII inhibition abolished PMA effects on wound closure (Fig. 2), suggesting a role for PKCβII in IEC wound closure in response to PMA activation. To confirm that the inhibition was specific to PKCβII we used an additional PKCβII specific inhibitor Enzastaurin (10 nM, [34]) and observed similar effects (data not shown). These findings were again corroborated in an additional IEC line, Caco2 (Figure S2). PMA treatment has been also shown to induce time dependent depletion of PKC isoforms [43]. Consistent with these findings we found that expression of PKCβII 16 hours after PMA treatment was significantly reduced (Figure S3A). This observation suggests that the reduced effect of PMA on wound repair at 24 hours compared to 12 hours (5.8±0.7 vs.15.9±2.6-fold increase at 24 h vs 12 h, respectively), was due to downregulation of PKCβII expression. Indeed, a decreased PMA-dependent effect on wound repair was evident at 16 hours following PMA treatment (8.3±1.2-fold increase, (Figure S3B).


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 PMA induced increase in wound closure.Confluent IEC monolayers were wounded by introduction of a single linear scratch wound, preincubated with the specified PKC inhibitors for 1 hour (Cheleretrine, pan PKC inhibitor (5 µM, pan); Calphostin C, Classical/non-conventional PKC inhibitor (10 µM, Clas/Non-C); KIC1-1, classical PKC inhibitor (5 µM, Clas); KIBI31-1, PKCβI inhibitor (1 µM, PKCβI); KIBII31-1 (5 µM, PKCβII); KIG31-1, PKCγ inhibitor (5 µM, PKCβγ) and stimulated with PMA (200nM). The area of the wound was measured at 12 and 24 hours. Inhibition of PKCβII, but not inhibition of other members of the classical PKCs significantly diminished PMA enhanced wound closure. N = 3 independent experiments. **significantly different from control (PMA alone, p<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569445&req=5

pone-0055775-g002: PKCβII mediates PMA induced increase in wound closure.Confluent IEC monolayers were wounded by introduction of a single linear scratch wound, preincubated with the specified PKC inhibitors for 1 hour (Cheleretrine, pan PKC inhibitor (5 µM, pan); Calphostin C, Classical/non-conventional PKC inhibitor (10 µM, Clas/Non-C); KIC1-1, classical PKC inhibitor (5 µM, Clas); KIBI31-1, PKCβI inhibitor (1 µM, PKCβI); KIBII31-1 (5 µM, PKCβII); KIG31-1, PKCγ inhibitor (5 µM, PKCβγ) and stimulated with PMA (200nM). The area of the wound was measured at 12 and 24 hours. Inhibition of PKCβII, but not inhibition of other members of the classical PKCs significantly diminished PMA enhanced wound closure. N = 3 independent experiments. **significantly different from control (PMA alone, p<0.01).
Mentions: PMA activates both classical and non-conventional PKC subfamilies [39]. Thus to examine which PKC subfamily member promotes PMA mediated wound closure we used a panel of broad PKC inhibitors including, the pan PKC inhibitor (Cheleretrine, 5 µM [40]), the inhibitor of classical/non-conventional PKC isoforms, Calphostin C (10 µM) [41] and pan classical PKC inhibitors KIC1-1(5 µM) [36] and Go6976 (0.5 µM) [42]. Since the major effect of PMA on IECs wound closure was observed within the first 24 hours (Fig. 1A) we determined the effect of specific PKC inhibitors at 12 h and 24 h post injury. As shown in Figure 2 pan PKC inhibition as well as inhibition of classical and non-conventional PKCs reversed the PMA-induced increase in T84 IECs wound closure. Importantly, inhibition of classical PKCs alone was sufficient to inhibit the PMA-induced increase in T84 cell wound closure (Fig. 2), confirming that member/s of the classical PKC subfamily are primarily responsible for the PMA effect. Next we used PKC isoform-specific peptide inhibitors for selected classical PKCs including PKCβI (KIBI31-1, 1 µM [37]), PKCβII, (KIBII31-1, 5 µM) and PKCγ (KIG31-1, 5 µM) to identify the specific PKC isoforms that mediate the PMA effect on IEC wound closure. PKCγ and PKCβI inhibition had no significant effect on PMA induced wound closure. However, in contrast, PKCβII inhibition abolished PMA effects on wound closure (Fig. 2), suggesting a role for PKCβII in IEC wound closure in response to PMA activation. To confirm that the inhibition was specific to PKCβII we used an additional PKCβII specific inhibitor Enzastaurin (10 nM, [34]) and observed similar effects (data not shown). These findings were again corroborated in an additional IEC line, Caco2 (Figure S2). PMA treatment has been also shown to induce time dependent depletion of PKC isoforms [43]. Consistent with these findings we found that expression of PKCβII 16 hours after PMA treatment was significantly reduced (Figure S3A). This observation suggests that the reduced effect of PMA on wound repair at 24 hours compared to 12 hours (5.8±0.7 vs.15.9±2.6-fold increase at 24 h vs 12 h, respectively), was due to downregulation of PKCβII expression. Indeed, a decreased PMA-dependent effect on wound repair was evident at 16 hours following PMA treatment (8.3±1.2-fold increase, (Figure S3B).

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