Hyperosmotic Stress-induced ATF-2 Activation through Polo-like Kinase 3 in Human Corneal Epithelial Cells

Ling Wang,Reid Payton,Wei Dai,Luo Lu

doi:10.1074/jbc.m110.166009

Abstract

Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation.

Highlights

Hyperosmotic stresses, resulting in delay of wound healing and development of dry eye and other eye diseases (8)
The effect of hyperosmotic stress-induced ATF-2 phosphorylation was studied by stimulating various human corneal epithelial cells with increased extracellular osmotic pressures that were created by adding high concentrations of sorbitol up to 1,000 mM
The effect of hyperosmotic stress-induced ATF-2 phosphorylation by 300 mM of glucose, sucrose, and NaCl were tested to compare with the effect of high concentration sorbitol in human corneal epithelial (HCE) cells (Fig. 1C)

Summary

The abbreviations used are

Polo-like kinase; HCE, human corneal epithelial; KD, kinase domain; CRE, cAMP response element; PP2, 4amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; ATM/ ATR, ataxia-telangiectasia mutated/ATM and Rad3-related. Composition, regulation, and function of AP-1 complex are different depending on the cellular context and activation of various MAPKs. In corneal epithelial cells, activation of JNK and p38 result in increase in cell mobility and apoptosis (27–30). We reported that Plk[3] is involved in UV irradiationand hypoxia-induced cell death by activation of c-Jun in cornea epithelial cells (16, 17). We investigated whether hyperosmotic stress induces Plk[3] activation, which subsequently alters corneal epithelial cell function through activation of ATF-2 in formation of AP-1 complex. Our results revealed that Plk[3] is a newly recognized component in signaling pathways to transmit extracellular hyperosmotic stress signals and to regulate ATF-2 in AP-1 complex in addition to the existing kinase cascade pathways

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION