Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Torrance, California 90502, USA.
Invest Ophthalmol Vis Sci. 2011 May 16;52(6):3200-6. doi: 10.1167/iovs.10-6485.
Hyperosmotic stress causes cell shrinkage, perturbs cell function, and damages DNA, resulting in cell cycle arrest and apoptosis. In the present study, the authors explore the mechanism involving hyperosmotic stress-induced activation of c-Jun/AP-1 through a novel Plk3 pathway in human corneal epithelial cells.
Human primary corneal epithelial cells and cell line were cultured in a serum-free keratinocyte medium and DMEM/F12 medium containing 10% FBS in a 37°C incubator supplied with 5% CO(2), respectively. Western blot analysis was used to determine protein expression and phosphorylation levels. Protein kinase activities were measured by immunocomplex kinase assay. Cell viability and apoptosis were determined by MTT assay and caspase-3 (DEVDase) activity.
It was found that hyperosmotic stress-induced increases in the phosphorylation of c-Jun, resulting in apoptosis through the activation of Plk3 in human corneal epithelial cells. Plk3 was activated by extracellular hyperosmotic stress to directly phosphorylate c-Jun in the serine 63 and 73 residues. Hyperosmotic stress-induced c-Jun phosphorylation was enhanced by overexpression of constitutively positive Plk3 mutants and suppressed by the knockdown of Plk3 mRNA with Plk3-specific siRNA. Further studies indicated that the phosphorylation of c-Jun by Plk3 was responsible for hyperosmotic stress-induced apoptosis, which was independent from activation of the JNK signaling pathway in human corneal epithelial cells.
These results, for the first time, provide a novel and alternative signaling mechanism that involves hyperosmotic stress-induced activation of the Plk3 pathway in addition to JNK/p38 MAPK pathways to regulate the c-Jun/AP-1 transcriptional complex and human corneal epithelial cell fate.
高渗应激导致细胞收缩，扰乱细胞功能，并损害 DNA，导致细胞周期停滞和细胞凋亡。本研究旨在探索人角膜上皮细胞中通过新型 Plk3 途径参与高渗应激诱导的 c-Jun/AP-1 激活的机制。
原代人角膜上皮细胞和细胞系分别在无血清角质形成细胞培养基和含 10% FBS 的 DMEM/F12 培养基中，于 37°C、5%CO2 孵育箱中培养。Western blot 分析用于确定蛋白表达和磷酸化水平。免疫复合物激酶测定法测定蛋白激酶活性。MTT 测定法和 caspase-3（DEVDase）活性用于测定细胞活力和细胞凋亡。
研究发现，高渗应激诱导 c-Jun 磷酸化增加，通过人角膜上皮细胞中 Plk3 的激活导致细胞凋亡。Plk3 被细胞外高渗应激激活，直接在丝氨酸 63 和 73 残基磷酸化 c-Jun。过表达组成型激活 Plk3 突变体增强了高渗应激诱导的 c-Jun 磷酸化，而 Plk3 特异性 siRNA 敲低 Plk3 mRNA 则抑制了 c-Jun 磷酸化。进一步的研究表明，Plk3 对 c-Jun 的磷酸化是高渗应激诱导的人角膜上皮细胞凋亡的原因，这与 JNK/p38 MAPK 途径的激活无关。
这些结果首次提供了一种新的替代信号机制，涉及高渗应激诱导的 Plk3 途径的激活，以及 JNK/p38 MAPK 途径，以调节 c-Jun/AP-1 转录复合物和人角膜上皮细胞的命运。
