Hyperosmotic stress-induced ATF-2 activation through Polo-like kinase 3 in human corneal epithelial cells

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.

FIGURE 1.

Hyperosmotic stress-induced ATF-2 phosphorylation through activation of Plk3. A, increases in ATF-2 phosphorylation following increased sorbitol concentrations in various human corneal epithelial cells. PHCE, primary HCE; HTCE, human telomerase-immortalized corneal epithelial. B, high concentration sorbitol-induced ATF-2 phosphorylation following a time course. Ctrl, control. C, effects of high concentrations of glucose (300 mm), sucrose (300 mm), and NaCl (300 mm) on ATF-2 phosphorylation. D, hyperosmotic dose-dependent activation of Plk3. E, effect of knocking down Plk3 mRNA on hyperosmotic stress-induced ATF-2 phosphorylation at 15 min. F, suppression of hyperosmotic stress-induced Plk3 activation by knocking down Plk3 mRNA with specific siRNA. Plk3 activity was determined by immunocomplex kinase assay, and ATF-2 fusion protein was served as the substrate. *, significant difference at p < 0.05 (n = 4).

FIGURE 2.

Activation of Plk3 by hyperosmotic stress resulting in ATF-2 phosphorylation. A, determination of phosphorylation site in ATF-2 catalyzed by purified and activated Plk3 kinase (Plk3a) fusion protein in vitro. B, activated Plk3a fusion protein failed to phosphorylate ATF-2 mutant (ATF-2^T69A/T71A), in which Thr-69 and Thr-71 were substituted by alanine residues. C, comparing the effect of activated Plk3a and kinase-defective Plk3^K52R fusion proteins on ATF-2 phosphorylation. D, comparing the effect of hyperosmotic stress-activated Plk3 in HCE cells on phosphorylation of ATF-2 and ATF-2^T69A/T71A. E, effect of hyperosmotic stress-activated Plk3^wt and Plk3^K52R on phosphorylation of ATF-2 in transfected HCE cells. *, a significant difference between Plk3^wt and Plk3^K52R transfected HCE cells (p < 0.05, n = 3).

FIGURE 3.

Effects of altered Plk3 activity on hyperosmotic stress-induced ATF-2 transcription activity. A, effects of overexpressing wild type Plk3 (pEGFP-Plk3-FL) and active Plk3 kinase domain (pEGFP-Plk3-KD) on hyperosmotic stress-induced ATF-2 phosphorylation. B, effects of overexpressing pEGFP-Plk3-FL and pEGFP-Plk3-KD on hyperosmotic stress-induced CRE reporter activity. C, effects of overexpressing pEGFP-Plk3-KD and pEGFP-Plk3^T219E mutant on hyperosmotic stress-induced CRE reporter activity. D, effects of overexpressing pEGFP-Plk3-FL and pEGFP-Plk3-KD on hyperosmotic stress-induced CRE reporter activity in ATF-2 or ATF-2^T69A/T71A cotransfected cells. * and **, significant differences between control and pEGFP-Plk3-FL/pEGFP-Plk3-KD transfected cells and between ATF-2/ATF-2^T69A/T71A cotransfected cells, respectively (p < 0.05, n = 6).

FIGURE 4.

Determining interaction between Plk3 and ATF-2 in hyperosmotic stress-induced cells. A, detection of Plk3 and ATF-2 interaction by immunoprecipitation (IP) using anti-phospho-ATF-2 (pAFT-2) antibody. Plk3 was detected by Western analysis. B, detection of Plk3 and ATF-2 interaction by immunoprecipitation using anti-Plk3 antibody. ATF-2 was detected by Western analysis using anti-pATF-2 antibody. C, detection of Plk3 distribution in the cytosolic and nuclear compartments with/without hyperosmotic stimulation. D, coimmunolocalization of Plk3 and ATF-2 in hyperosmotic stress-induced cells. E, high amplification view (60×) to show colocalization of Plk3 and phospho-ATF-2 in the nucleus of the hyperosmotic stress-induced cell. Coimmunolocalization experiments were done by immunostaining and double staining of hyperosmotic stress-induced human corneal epithelial cells using antibodies specific to Plk3, ATF-2, and phospho-ATF-2. Photo images were taken by using a Nikon fluorescent microscope at 20× and 60×, respectively. The data were analyzed with a Nikon software program.

FIGURE 5.

Effects of suppressing JNK/p38 on hyperosmotic stress-induced Plk3 activation. A, effect of knocking down JNK or p38 mRNAs on hyperosmotic stress-induced Plk3 expression and ATF-2 activation. B, effect of knocking down JNK or p38 mRNAs on hyperosmotic stress-induced Plk3 activity. C, effect of knocking down Plk3 mRNA on hyperosmotic stress-induced JNK and p38 phosphorylation. D, effect of knocking down Plk3 mRNA on hyperosmotic stress-induced JNK and p38 activities. Phosphorylation of JNK/p38 was measured by using anti-phospho-JNK and anti-phospho-p38 antibodies, respectively. The activities of JNK, p38, and Plk3 were determined in three sets of independent experiments by immunocomplex kinase assays, and ATF-2 fusion protein was used as the substrate.

FIGURE 6.

Differentiation of Plk3 effect from JNK and p38 on ATF-2 phosphorylation in hyperosmotic stress-induced cells. A, effect of knocking down JNK mRNA on hyperosmotic stress-induced ATF-2 phosphorylation. B, effect of knocking down p38 mRNA on hyperosmotic stress-induced ATF-2 phosphorylation. C, effects of blocking JNK and p38 with SP600125 and SB202190, respectively, on hyperosmotic stress-induced ATF-2 phosphorylation. D, effects of knocking down Plk3 in the absence and presence of SP600125 and SB202190 on hyperosmotic stress-induced ATF-2 phosphorylation. E, effect of hyperosmotic stress on Plk3 activity. Plk3 activity was determined by immunocomplex kinase assay, and ATF-2 fusion protein was used as the substrate. An immunodepletion procedure was used to remove JNK and p38 in cell lysates. F, effects of high concentrations of high concentrations of glucose (300 mm), sucrose (300 mm), and NaCl (300 mm) on Plk3 activities. *, a significant difference between control and hyperosmotic stress-induced cells (p < 0.05, n = 3).

FIGURE 7.

Effect of inhibiting Src and ATM/ATR on hyperosmotic stress-induced Plk3 and ATF-2 activation. A, effect of inhibiting Src with PP2 on hyperosmotic stress-induced ATF-2 phosphorylation. B, effect of inhibiting Src with PP2 on hyperosmotic stress-induced Plk3 activity. C, effect of inhibiting ATM/ATR with caffeine (Caff) on hyperosmotic stress-induced ATF-2 phosphorylation. D, effect of inhibiting ATM/ATR with caffeine on hyperosmotic stress-induced Plk3 activity. Phosphorylation of ATF-2 was measured by Western analysis using anti-phospho-ATF-2 (at site of threonine 71) antibody. Plk3 activity was determined in three sets of independent experiments by immunocomplex kinase assays.