Vitamin C Is a Kinase Inhibitor: Dehydroascorbic Acid Inhibits IκBα Kinase β

Dietary vitamin C is essential for humans, primates, guinea pigs, and several other animals and insects that lack l-gulono-γ-lactone oxidase, the final enzyme in its biosynthetic pathway from glucose (25). Under physiological conditions, vitamin C predominantly exists in its reduced form, ascorbic acid (AA); it also exists in trace quantities in the oxidized form, dehydroascorbic acid (DHA). There are two known mechanisms for transporting vitamin C (21). A universal system, present in all cells, transports vitamin C as DHA via facilitative glucose transporters (34). Once inside the cell, DHA is rapidly reduced and accumulates as AA (34, 35). The second transport system is functional in specialized cells where AA is directly transported into cells via sodium-dependent AA cotransporters (SVCT1 and/or SVCT2) (33). AA functions as a cofactor for enzymes involved in the biosynthesis of collagen (26), carnitine (28), and norepinephrine (18) and in the amidation of hormones (8). In plasma and cells, AA is a powerful antioxidant, quenching reactive oxygen species (ROS) and reactive nitrogen species (10, 14). Intracellular vitamin C can prevent cell death and inhibit mutations induced by oxidative stress (12, 22, 37). During the process of quenching free radicals, ascorbate donates an electron, becoming the unstable intermediate ascorbyl radical that can be reversibly reduced back to ascorbate. Ascorbyl radical can donate a second electron and be converted to DHA (13, 14). DHA may be reduced back to AA or be irreversibly hydrolyzed to 2,3-diketo-gulonic acid, which then is metabolized to threonic and oxalic acid (14). In cells loaded with AA and exposed to hydrogen peroxide, AA is converted to DHA, some of which effluxes from the cells via the glucose transporters, thereby providing a mechanism for recycling vitamin C to the extracellular medium (12). Alternatively, intracellular DHA can be transported to intracellular compartments and organelles (2, 20). DHA functions primarily as a readily transportable form of vitamin C (36). ROS play a key role in cellular responses as chemical second messenger molecules, and conversely, antioxidants modulate selected signaling responses (24). For example, ROS activate transcription factors, such as NF-κB, that are important in host defense, inflammation, and apoptosis (1, 11, 32). Pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), hydrogen peroxide, and ceramide, activate NF-κB by inducing the phosphorylation of IκB proteins (11, 19). Phosphorylated IκBα releases NF-κB and is itself degraded via proteasomal pathways (17), while unphosphorylated IκB associates with NF-κB in the cytosol, preventing its nuclear migration. It was initially reported that AA inhibits TNF-α-induced NF-κB activation in endothelial cells via activation of p38 mitogen-activated protein kinase (MAPK) (4); however, we recently showed that AA suppresses TNF-α-dependent activation of NF-κB by inhibiting the activation of kinases involved in the phosphorylation of IκBα (6). We investigated the modulation of NF-κB activation by vitamin C and found that DHA directly inhibited the kinase activity of IKKβ and IKKα in vitro and in cellular assays. Thus, our data suggest a dual mechanisms of action of vitamin C in regulating NF-κB function. First, as an antioxidant quenching ROS, AA inhibits ROS-mediated signaling events. Second, after oxidization to DHA, vitamin C directly inhibits IKK kinase activity.