Hyperosmotic stress-induced corneal epithelial cell death through activation of Polo-like kinase 3 and c-Jun.

The corneal epithelial layer in the front of the eye forms the first line of defense to protect the structures behind the cornea from the insults and injuries of environmental hazards, including hyperosmotic stress.1 Changes in extracellular osmotic pressure are often found in some pathologic conditions, such as diabetes mellitus, uremia, dehydration after exercise, heat shock, fatal burns, and inflammation sites in various tissues, including the cornea.2–5 In the cornea, hyperosmotic stress from environmental and pathologic conditions strikes the epithelial cells and alters the fluid balance, resulting in cell shrinkage, which may contribute to delayed wound healing and dry eye diseases.6 Hyperosmotic stress extracts water from the cell, inducing cell shrinkage. To restore the volume, cells undergo a process of regulated volume increase within several minutes by the uptake of inorganic ions and water.7–9 Cell shrinkage and increased ionic strength alternate cell architecture compartments, denature proteins, and disturb cell function.10–12 It has been shown that persistent hyperosmotic stress can induce DNA damage, cell cycle arrest, and apoptosis.11,13 Hyperosmotic stress induces a series of changes in intracellular kinase cascades that include the activation of JNK, p38, and other signaling pathways to activate the important AP-1 (activating protein-1) transcription factor complex.12,14–16 AP-1 acts as a central switch, in addition to the NF-κB and NFAT5 pathway, which has been known as a primary pathway in response to osmotic stress, to convert extracellular signals into genetic responses by regulating stress-related gene expression to determine cell proliferation, differentiation, and apoptosis.17,18 One of the important hyperosmotic stress-activated transcription factors is c-Jun, which participates in the formation of AP-1 dimers with a group of the other transcription factors, such as members of the Jun, Fos, and ATF families.19,20 In response to stress stimulation, c-Jun is activated mainly through the JNK signaling pathway in various cell types, including corneal epithelial cells. In addition, activation of the JNK signaling pathway by stress stimulation results in increased cell mobility and apoptosis.21–24 In addition, hyperosmotic stress can induce the production of certain corneal epithelial proteins, resulting in decreased cell viability through JNK MAPK-mediated pathways.25 In our previous studies, we found that c-Jun can also be activated by UV irradiation and hypoxia/reoxygenation stress through activation of the Polo-like kinase 3 (Plk3) signaling pathway in corneal epithelial cells.26,27 In mammalian cells, Plk3 belongs to a family consisting of four members (Plk1, Plk2, Plk3, Plk4) that share highly conserved homologies to Drosophila Polo kinase.28–31 All Plk family members contain a kinase domain (KD) at the N terminus that phosphorylates downstream proteins at serine/threonine residues and a Polo-box domain (PBD) at the C terminus that binds to interactive motifs in the target proteins. As a multifunctional protein, Plk3 is involved in regulating a variety of molecular and intracellular events, including DNA damage response, cell cycle control, and apoptosis.32,33 Plk3 undergoes substantial changes in its kinase activity and subcellular distribution after cell cycle progression. Plk3 is rapidly activated on stress stimulation by ionizing radiation, reactive oxygen species, methylmethane sulfonate, UV irradiation, and hypoxia.26,27,34 In addition, hypoxic stress-induced delay of corneal epithelial wound healing is significantly improved in Plk3-deficient mice, indicating that Plk3 plays an important role in the wound healing process.26 However, it is still unclear in hyperosmotic stress-stimulated cells whether Plk3 is activated and how active Plk3 phosphorylates c-Jun to regulate cell function. In the present study, we report that Plk3 is involved in hyperosmotic stress-induced cell death through the phosphorylation of c-Jun protein to activate c-Jun in corneal epithelial cells. Our results reveal that Plk3 plays an important role in the signaling cascades to transmit extracellular hyperosmotic stress signals to the regulation of c-Jun in the AP-1 transcription complex in addition to the known JNK signaling pathway, which has been recognized to connect hyperosmotic stress stimulation to cell fate.