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.
Highlights
Intestinal epithelial cells (IECs) form an important barrier that separates luminal contents from underlying tissue compartments
Since epithelial wound healing requires cell migration and proliferation, we hypothesized that Protein kinase C (PKC) activation promotes IEC wound closure
Since we determined that phorbol 12-myristate 13-acetate (PMA) enhanced wound closure was primarily mediated by PKCbII, we examined the cellular localization of PKCbII, as well as other classical PKCs, PKCbI and PKCc in T84 IECs stimulated with PMA for 4 hours
Summary
Intestinal epithelial cells (IECs) form an important barrier that separates luminal contents from underlying tissue compartments. Epithelial wounds secondary to inflammation and ischemia rapidly reseal to re-establish this critical barrier [1]. Epithelial wound healing is typically mediated by cell migration and proliferation [2]. Small wounds reseal efficiently by cell migration alone, and this process is referred to as wound restitution. Spreading epithelial cells polarize to reorient their microtubule organizing center (MTOC) and Golgi apparatus in the direction of the wound [6], as well as extend cellular membrane protrusions at the leading edge referred to as lamellipodia which play an important role in mediating forward cell movement [7,8]. Cell movement requires dynamic turnover of focal cell matrix associations and restructuring of the actin cytoskeleton [9]
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