Abstract
Reactive oxygen species of mitochondrial origin have been implicated in regulating the expression of several tumor necrosis factor (TNF)-induced genes. Manganese superoxide dismutase (Mn-SOD) is one of many genes, but only antioxidant enzyme, induced in response to tumor necrosis factor. Mn-SOD is a nuclear-encoded mitochondrial matrix protein and serves a protective function by detoxifying superoxide. To address the role of superoxide in regulating gene expression in response to TNF, we have constitutively overexpressed Mn-SOD in a human fibrosarcoma cell line and asked what effect this has on the expression of a number of TNF-responsive genes using reverse transcription-polymerase chain reaction. Of the TNF-induced transcripts analyzed, only interleukin-1alpha (IL-1alpha) was modulated in response to Mn-SOD overexpression. In all cases of Mn-SOD overexpression, IL-1alpha protein and mRNA levels were lowered constitutively and in response to TNF when compared to the parental and mock-transfected cell lines. The induction of IL-1alpha by TNF can also be decreased by growth in 3% oxygen as compared to growth in 21% O2; in addition, growth in low oxygen lowers the basal level of IL-1alpha protein. The effect of Mn-SOD overexpression on IL-1alpha expression can be overcome by treatment with the protein kinase C activator, phorbol 12-myristate 13-acetate. Mn-SOD overexpression and low oxygen alter IL-1alpha mRNA levels by decreasing the stability of the IL-1alpha mRNA. These findings indicate that both Mn-SOD and O2 may regulate the levels of a cellular oxidant involved in both basal and TNF-induced IL-1alpha expression, presumably superoxide.
Highlights
Manganese superoxide dismutase (Mn-SOD)1 is one of three superoxide dismutases found in mammalian cells which catalyze the dismutation reaction of superoxide to hydrogen peroxide [1]
Our findings suggest that Mn-SOD plays a role in the tumor necrosis factor (TNF) signal pathway leading to increased IL-1␣ expression
The Mn-SOD overexpression in contrast to PKC inhibitors does not block but abates the increase in IL-1␣ and suggests that both PKC activation and mitochondrial reactive oxygen species may be required for the maximal increase in IL-1␣ levels in response to TNF
Summary
Mn-SOD, manganese superoxide dismutase; TNF, tumor necrosis factor; GAPDH, glyceraldehyde phosphate dehydrogenase, PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; IL, interleukin; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; LPS, lipopolysaccharide; CAT, chloramphenicol acetyltransferase; TEMED, N,N,NЈ,NЈ-tetramethylethylenediamine. Exposure to high levels of reactive oxygen species is toxic to all cells, but lower levels cause extensive adaptive responses, including greatly enhanced resistance [18, 19] Reactive oxygen species, such as superoxide and hydrogen peroxide, have been proposed to play a role in cellular signal-transduction [20, 21]. The pathway leading to TNF-mediated activation of NFB has been shown to require oxygen-derived radicals of mitochondrial origin [25, 26] These findings prompted us to investigate what role Mn-SOD may play in regulating the levels of these oxygen-derived signal molecules of mitochondrial origin
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