TY - JOUR TI - Responses of murine keratinocytes to oxidative stress DO - https://doi.org/doi:10.7282/T36W9BJN PY - 2007 AB - Oxidative stress is well recognized as a major contributing factor in the development of cutaneous disease. The generation of reactive oxygen intermediates (ROI) damage cellular macromolecules, causing mutagenic DNA lesions, altered enzyme function, lipid peroxidation and inappropriate signaling. Induction of oxidative stress is associated with the onset of inflammation through production of arachidonic acid-derived lipid mediators including prostaglandins and leukotrienes although the mechanisms linking these two processes are unclear at this time. As the protective barrier of the body, the skin is exposed to many oxidants. Two such potent inducers of oxidative stress are the high energy wavelengths of the ultraviolet B (UVB) spectra (290-320 nm) and the redoxcycling herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium). In the present studies, we compared the effect of UVB and paraquat on the expression of antioxidant and arachidonic acid metabolism enzymes in undifferentiated and calcium-differentiated primary mouse keratinocytes. Using enzyme assays, we found that both UVB (2.5-25mJ/cm2) and paraquat (100 µM) generated ROI and were effective inducers of oxidative stress in these cells and that paraquat readily undergoes redox-cycling. Both agents upregulated the expression of several antioxidant enzymes, measured using realtime PCR although a greater number of enzymes were induced by paraquat. UVB and paraquat equally upregulated eicosanoid enzyme expression; these enzymes included the cyclooxygenases, prostanoid synthases, lipoxygenases and leukotriene synthetic enzymes as well as the prostaglandin and leukotriene receptors. Using HPLC with fluorescence detection, UVB was found to modulate prostaglandin production. In both cell types, UVB caused a dose-dependent activation of the p38 and JNK MAP kinases. Akt kinase, however, was found to be activated only in undifferentiated cells with constitutive phosphorylation in differentiated cells. Inhibition of these enzymes markedly inhibited mRNA expression of several eicosanoid enzymes while other enzymes other were unaffected. Taken together, these data demonstrate that UVB light and paraquat effectively induce both antioxidant and eicosanoid biosynthetic enzymes in mouse keratinocytes and that activation of these pathways are regulated by MAP and Akt kinase activity. The elucidation of the keratinocyte response to oxidative stress is a necessary step to a better understanding of the effects of oxidants on the skin. KW - Toxicology KW - Keratinocytes KW - Mice LA - English ER -