Abstract
Purpose: :
CAP37, an inflammatory mediator released from neutrophils, is a known chemotactic agent for human corneal epithelial cells (HCEC). The focus of this study was to determine the signaling pathway that regulates CAP37-dependent HCEC chemotaxis.
Methods: :
HCECs were treated with inhibitors of protein kinase C (PKC, calphostin-C, bis-indolylmaleimide, and Ro 31-8220), protein kinase A (PKA, H-89), JNK (JNK inhibitor II), MEK1 (PD98059), PDBu (4β-Phorbol 12, 13-dibutyrate), or siRNA, and the effect on migration in response to recombinant CAP37 (rCAP37) was measured using a modified Boyden chemotaxis chamber. Constitutive expression of PKC isoforms, PKC depletion after PDBu treatment, identification of subcellular localization of PKC isoforms, and siRNA treatment against PKC Δ or θ in HCECs was determined by Western blot analysis of HCEC cell lysates. PKC translocation or inhibition of translocation with rabbit antiserum to CAP37 was visualized using confocal microscopy with primary antibodies to PKC α, Δ, ε, and θ followed by Alexa488 secondary antibodies.
Results: :
Pharmacological inhibitors of PKC significantly decreased chemotaxis of HCECs in response to rCAP37, while inhibitors of PKA, JNK, and MEK1 had a negligible effect on CAP37-dependent chemotaxis. CAP37-directed migration was also inhibited following PKC depletion with PDBu. HCEC constitutively expressed all isoforms of PKC except PKC β and γ, however only PKC α, Δ, ε, and θ were sensitive to PDBu depletion. Confocal microscopy showed that PKC Δ and θ changed their cellular localization after treatment with rCAP37 in a time- and dose-dependent manner. This response was inhibited with antiserum to CAP37. Subcellular analysis of CAP37-treated HCECs showed movement of PKC Δ and θ to the membrane fraction when compared to untreated samples, verifying the confocal observations. Knocking-down PKC Δ and θ resulted in a significant decrease in CAP37-mediated migration.
Conclusions: :
These data suggest that CAP37-dependent HCEC chemotaxis may be governed by the PKC signaling pathway specifically through the PKC Δ and/or θ isoforms.
Keywords: cornea: epithelium • signal transduction • wound healing