Chotiros Daosukho

Identification of Nucleophosmin as an NF-κB Co-activator for the Induction of the Human SOD2 Gene *

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-κB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-κB affinity chromatography to isolate potential NF-κB interacting proteins. Proteins eluted from the NF-κB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-κB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-κB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines.

NF-κB-Associated MnSOD induction protects against β-amyloid-induced neuronal apoptosis

Expression of manganese superoxide dismutase (MnSOD), a nuclear-encoded mitochondrial primary antioxidant enzyme, is protective against various paradigms of oxidative stress-induced brain injury. We have shown previously that the presence of an intronic nuclear factor site, κB (NF-κB), in the MnSOD gene is essential for the induction of MnSOD by tumor necrosis factor α (TNF-α). However, whether activation of NF-κB is protective against oxidative stress-induced neuronal injury is unclear. In the present study, we demonstrate that TNF-α activates NF-κB activity in neuronal, SH-SY5Y, cells and preferentially enhances the binding of p50 and p65 to the promoter/enhancer regions of the MnSOD gene. Binding of NF-κB members to the MnSOD gene leads to the induction of MnSOD mRNA and protein levels. Consequently, induction of MnSOD by TNF-α primes neuronal cells to develop resistance against subsequent exposure to β-amyloid and FeSO4. Taken together, these results suggest that NF-κB might exert its protective function by induction of MnSOD leading to subsequent protection against oxidative stress-induced neuronal injury.

Tumor necrosis factor receptor deficiency exacerbated Adriamycin-induced cardiomyocytes apoptosis: an insight into the Fas connection

Cardiomyopathy is a major dose-limiting factor for applications of Adriamycin, a potent chemotherapeutic agent. The present study tested the hypothesis that increased tumor necrosis factor (TNF)-α signaling via its receptors protects against Adriamycin-induced cardiac injury. We used mice in which both TNF receptor I and II have been selectively inactivated (DKO) with wild-type mice as controls. Morphometric studies of cardiac tissue following Adriamycin treatment revealed greater ultrastructural damage in cardiomyocyte mitochondria from DKO mice. Biochemical studies of cardiac tissues showed cytochrome c release and the increase in proapoptotic protein levels, suggesting that lack of TNF-α receptor I and II exacerbates Adriamycin-induced cardiac injury. The protective role of TNF receptor I and II was directly confirmed in isolated primary cardiomyocytes. Interestingly, following Adriamycin treatment, the levels of Fas decreased in the wild-type mice. In contrast, DKO mice had an increase in Fas levels and its downstream target, mitochondrial truncated Bid. These results suggested that TNF-α receptors play a critical role in cardioprotection by suppression of the mitochondrial-mediated associated cell death pathway. [Mol Cancer Ther 2006;5(2):261–9]

Tamoxifen enhancement of TNF-α induced MnSOD expression: modulation of NF-κB dimerization

Manganese superoxide dismutase (MnSOD) has been shown to suppress the development of cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen that is widely used in chemotherapy, is known to be a modulator of antioxidant status. However, the mechanism by which TAM mediates antioxidant enzyme induction remains unclear. In this study we investigated TAM enhancement of MnSOD induction by TNF-α. The results show that co-treatment with TAM and TNF-α increases the MnSOD promoter/enhancer driven luciferase activity, MnSOD mRNA and protein levels. Interestingly, co-treatment with TAM and TNF-α drastically decreases the binding activity of the p50/p50 homodimer and increases that of the p50/p65 heterodimer compared to TNF-α alone. This change in DNA binding could not be attributed to a decrease in the level of p50, its precursor, p105, or its inhibitors. Furthermore, TAM did not enhance degradation of IκB-α. These results suggest that p50/p50 homodimer may act as an inhibitory complex of MnSOD expression. Modulation of the DNA binding activity in favor of the p50/p65 complex may enhance NF-κB mediated induction of MnSOD by TAM. These findings reveal a potential novel mechanism for the induction of the human MnSOD gene.

IκBα (inhibitory κBα) identified as labile repressor of MnSOD (manganese superoxide dismutase) expression

Cytokines, phorbol esters, radiation and chemotherapeutic drugs up-regulate the expression of MnSOD (manganese superoxide dismutase). Using the VA-13 cell line, we studied the regulation of SOD2 upon treatment with PMA. Pre-treatment with CHX (cycloheximide) followed by PMA led to significantly higher levels of MnSOD mRNA compared with those with either agent alone, suggesting de novo synthesis of an inhibitory protein. PMA treatment modulates redox-sensitive transcription factors, therefore we evaluated the effects of this combination treatment upon AP-1 (activator protein 1) and NF-κB (nuclear factor κB), two trans-acting factors suggested to play a role in SOD2 regulation. Co-administration of CHX and PMA led to a time-dependent increase in the binding activity of NF-κB. Therefore we evaluated IκBα (inhibitory κBα) and found that co-administration decreased its steady-state level compared with either agent alone, suggesting that enhanced NF-κB activation is due to inhibition of IκBα synthesis. PMA activates PKC (protein kinase C) enzymes which phosphorylate IκBα, leading to its degradation, therefore we used GF109203X to inhibit PKC activity. Stable transfection utilizing a PMA-responsive element in the human SOD2 gene, showed a concentration-dependent decrease in luciferase and NF-κB-binding activity with GF109203X. Western blot analysis indicated the presence of several PKC isoforms in the VA-13 cell line; however, PMA pre-treatment specifically down-regulated α and βI, suggesting a role for one or more of these proteins in SOD2 induction. Taken together, these results indicate that the PKC pathway leading to SOD2 induction proceeds at least in part through NF-κB and that inhibition of IκBα synthesis might serve as a potential pharmacological approach to up-regulate MnSOD.