Reactive oxygen species mediate UV-induced GSK-3β activation and p21 protein degradation

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Previously, we have reported that UV induced GSK-3β activation and subsequent proteasomal degradation of p21 protein. Here, the evidence is provided that UV-induced reactive oxygen species (ROS) generation causes activation of GSK-3β. ROS include superoxide anion (O₂.-), hydroxyl radicals (.OH), and hydrogen peroxide (H₂O₂) and it their production is enhanced when cells are exposed to various stimuli such as UV, ionizing radiation, cancer chemotherapeutics, and TNF-α. In a cell, ROS are generated in the cell as normal by-products of oxygen metabolism or by the activities of nicotinamade adenine dinucleotide phosphate-oxidase (NAD(P)H oxidase, NOX enzymes), cyclooxygenase, lipoxygenase and xanthine oxidase. ROS are capable of damaging cellular macromolecules including DNA, proteins, and lipids. In SK-MEL-2 and MCF-7 cells, ROS were produced as soon as 10 min after UV-irradiation as shown by DCFDA staining. Since it has been reported that GSK-3β can be activated by ROS, it was next determined whether ROS produced by UV-irradiation leaded to GSK-3β activation. When cells were pretreated with a ROS scavenger, N-acetyl-cysteine (NAC), UV-induced GSK-3β activation was inhibited, indicating that GSK-3β activation was mediated by ROS. NAC pretreatment also abolished p21 protein degradation after UV-irradiation. Furthermore, hydrogen peroxide induced GSK-3β activation. It was further shown that NADPH oxidase was responsible for the UV-induced ROS generation. NADPH oxidase is an enzyme complex which is assembled in the membranes upon activation. It generates superoxide by transferring electrons from NADPH to molecular oxygen. NADPH oxidase inhibitors, diphenylene iodonium (DPI) and apocynin, prevented ROS generation as well as GSK-3β activation after UV-irradiation. However, a GSK-3β inhibitor,4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD8) did not abolish ROS generation confirming that ROS generation was an upstream event to the GSK-3β activation. In summary, UV-induced GSK-3β activation is mediated by ROS and results in degradation of p21 protein.