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Uniform TitleResponses of murine keratinocytes to oxidative stress
NameBlack, Adrienne T. (author), Laskin, Jeffrey (chair), Laskin, Debra (internal member), Gerecke, Donald (internal member), Gordon, Marion (internal member), Heck, Diane (internal member), Heindel, Ned (outside member), Rutgers University, Graduate School-New Brunswick,
Degree Date2007
Date Created2007
SubjectToxicology,
Keratinocytes,
Mice
DescriptionOxidative 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.
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.
Note[degree] Ph.D.
Genretheses
Persistent URLhttp://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.13490
LanguageEnglish
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.