Glutathione supplementation potentiates hypoxic apoptosis by S-glutathionylation of p65-NFkappaB

In murine embryonic fibroblasts, N-acetyl-L-cysteine (NAC), a GSH generating agent, enhances hypoxic apoptosis by blocking the NFkappaB survival pathway (Qanungo, S., Wang, M., and Nieminen, A. L. (2004) J. Biol. Chem. 279, 50455-50464). Here, we examined sulfhydryl modifications of the p65 subunit of NFkappaB that are responsible for NFkappaB inactivation. In MIA PaCa-2 pancreatic cancer cells, hypoxia increased p65-NFkappaB DNA binding and NFkappaB transactivation by 2.6- and 2.8-fold, respectively. NAC blocked these events without having an effect on p65-NFkappaB protein levels and p65-NFkappaB nuclear translocation during hypoxia. Pharmacological inhibition of the NFkappaB pathway also induced hypoxic apoptosis, indicating that the NFkappaB signaling pathway is a major protective mechanism against hypoxic apoptosis. In cell lysates after hypoxia and treatment with N-ethylmaleimide (thiol alkylating agent), dithiothreitol (disulfide reducing agent) was not able to increase binding of p65-NFkappaB to DNA, suggesting that most sulfhydryls in p65-NFkappaB protein were in reduced and activated forms after hypoxia, thereby being blocked by N-ethylmaleimide. In contrast, with hypoxic cells that were also treated with NAC, dithiothreitol increased p65-NFkappaB DNA binding. Glutaredoxin (GRx), which specifically catalyzes reduction of protein-SSG mixed disulfides, reversed inhibition of p65-NFkappaB DNA binding in extracts from cells treated with hypoxia plus NAC and restored NFkappaB activity. This finding indicated that p65-NFkappaB-SSG was formed in situ under hypoxia plus NAC conditions. In cells, knock-down of endogenous GRx1, which also promotes protein glutathionylation under hypoxic radical generating conditions, prevented NAC-induced NFkappaB inactivation and hypoxic apoptosis. The results indicate that GRx-dependent S-glutathionylation of p65-NFkappaB is most likely responsible for NAC-mediated NFkappaB inactivation and enhanced hypoxic apoptosis.     

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.     

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

Conjugated linoleic acid promotes human adipocyte insulin resistance through NFkappaB-dependent cytokine production

We previously demonstrated that trans-10, cis-12 conjugated linoleic acid (CLA) reduced the triglyceride content of human adipocytes by activating mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling via interleukins (IL) 6 and 8. However, the upstream mechanism is unknown. Here we show that CLA increased (>or=6 h) the secretion of IL-6 and IL-8 in cultures containing both differentiated adipocytes and stromal vascular (SV) cells, non-differentiated SV cells, and adipose tissue explants. CLA isomer-specific induction of IL-6 and tumor necrosis factor-alpha was associated with the activation of nuclear factor kappaB (NFkappaB) as evidenced by 1) phosphorylation of IkappaBalpha, IkappaBalpha kinase, and NFkappaB p65, 2) IkappaBalpha degradation, and 3) nuclear translocation of NFkappaB. Pretreatment with selective NFkappaB inhibitors and the MEK/ERK inhibitor U0126 blocked CLA-mediated IL-6 gene expression. Trans-10, cis-12 CLA suppression of insulin-stimulated glucose uptake at 24 h was associated with decreased total and plasma membrane glucose transporter 4 proteins. Inhibition of NFkappaB activation or depletion of NFkappaB by RNA interference using small interfering NFkappaB p65 attenuated CLA suppression of glucose transporter 4 and peroxisome proliferator-activated receptor gamma proteins and glucose uptake. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA promotes NFkappaB activation and subsequent induction of IL-6, which are at least in part responsible for trans-10, cis-12 CLA-mediated suppression of peroxisome proliferator-activated receptor gamma target gene expression and insulin sensitivity in mature human adipocytes.