Vitamin K prevents oxidative cell death by inhibiting activation of 12-lipoxygenase in developing oligodendrocytes

Abstract

Oxidative mechanisms of injury are important in many neurological disorders. Developing oligodendrocytes (pre-OLs) are particularly sensitive to oxidative stress-mediated injury. We previously demonstrated a novel function of phylloquinone (vitamin K(1)) and menaquinone 4 (MK-4; a major form of vitamin K2) in protecting pre-OLs and immature neurons against glutathione depletion-induced oxidative damage (Li et al. [ 2003] J. Neurosci. 23:5816-5826). Here we report that vitamin K at nanomolar concentrations prevents arachidonic acid-induced oxidative injury to pre-OLs through blocking the activation of 12-lipoxygenase (12-LOX). Arachidonic acid metabolism is a potential source for reactive oxygen species (ROS) generation during ischemia and reperfusion. Exposure of pre-OLs to arachidonic acid resulted in oxidative cell death in a concentration-dependent manner. Administration of vitamin K (K(1) and MK-4) completely prevented the toxicity. Consistent with our previous findings, inhibitors of 12-LOX abolished ROS production and cell death, indicating that activation of 12-LOX is a key event in arachidonic acid-induced pre-OL death. Vitamin K(1) and MK-4 significantly blocked 12-LOX activation and prevented ROS accumulation in pre-OLs challenged with arachidonic acid. However, vitamin K itself did not directly inhibit 12-LOX enzymatic activity when assayed with purified 12-LOX in vitro. These results suggest that vitamin K, or likely its metabolites, acts upstream of activation of 12-LOX in pre-OLs. In summary, our data indicate that vitamin K prevents oxidative cell death by blocking activation of 12-LOX and ROS generation.

Fig. 1

Arachidonic acid induces oxidative death of pre-OLs. A: Arachidonic acid (AA) induced loss of pre-OL viability in a concentration-dependent manner. Data represent mean ± SEM of six independent experiments. B: Antioxidants vitamin E (0.1 μM) and coenzyme Q₄ (1 μM) completely abolished the arachidonic acid-induced pre-OL death. Data are representative of four independent experiments. C: Reactive oxygen species were generated in pre-OLs challenged with arachidonic acid (100 μM). Data represent two separate experiments. D: The pan-caspase inhibitor z-VAD-fmk did not prevent arachidonic acid-induced cell death. Pre-OLs were treated with arachidonic acid (60 μM) in the presence or absence of z-VAD-fmk (50 μM) for 24 hr, and cell viability was analyzed. Data are representative of three separate experiments.

Fig. 2

Vitamin K₁ and menaquinone-4 completely abolish oxidative pre-OL death. A: Vitamin K₁ and MK-4 prevent arachidonic acid-induced pre-OL death in a concentration-dependent manner. Pre-OLs were challenged with arachidonic acid (100 μM) in the presence of increasing concentrations of K₁ or MK-4 for 24 hr, and cell survival was determined as described. B: Representative phase-contrast images of pre-OLs treated as indicated for 20–24 hr. Pre-OLs were treated with arachidonic acid (100 μM) in the absence or presence of K₁ (1 μM) and MK-4 (0.1 μM) as indicated. Data are representative of at least three experiments. Scale bar = 50 μm.

Fig. 3

Vitamin K₁ and menaquinone-4 prevent arachidonic acid-induced ROS accumulation in pre-OLs. Pre-OLs were treated as indicated for 18–20 hr, and ROS accumulation was visualized with dihydrorhodamine 123 (A) and quantified with DCFH-DA (B). The concentrations of K₁, MK-4, and CoQ₄ were 1 μM. DCFH-DA and dihydrorhodamine are nonfluorescent but become fluorescent upon oxidation by peroxides generated in cells. Data are representative of two separate experiments. Scale bar = 50 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4

Vitamin K₁ and menaquinone-4 inhibit arachidonic acid-induced 12-lipoxygenase activation. A,B: The 12-LOX inhibitor AA861 prevented arachidonic acid-induced pre-OL death and ROS accumulation. Pre-OLs were treated as indicated with or without arachidonic acid (200 μM) in the absence or presence of AA861 (10 μM). Cell viability and ROS generation were evaluated 24 and 20 hr later, respectively. C: The 12-LOX-specific inhibitor AA861 blocked arachidonic acid-induced activation of 12-LOX and accumulation of its product 12-HETE. **P < 0.01 compared with controls. D: Vitamin K₁ and MK-4 prevented arachidonic acid-induced 12-HETE accumulation in pre-OLs. Data are representative of three experiments. *P < 0.05, **P < 0.001 vs. the controls.

Fig. 5

Vitamin K₁ and menaquinone-4 do not directly inhibit 12-lipoxygenase enzymatic activity in a cell-free system. A: Purified 12-LOX (20 U) was incubated with AA861 (10 μM) or MK-4 (10 μM) for 30 min prior to addition of the 12-LOX substrate arachidonic acid (100 μM). The reaction mix was incubated for 20 min at 37°C. 12-HETE was extracted and analyzed by thin-layer chromatography as described. Positions of origins of sample application, 12-HETE, and free arachidonic acid are indicated by arrows. B: The 12-LOX-specific inhibitor BHPP (2–20 μM) dose dependently blocked 12-HETE production. Data are from one experiment representative of three that were performed.