beta-D-Glucoside suppresses tumor necrosis factor-induced activation of nuclear transcription factor kappaB but potentiates apoptosis

Mangiferin, a natural polyphenol is known to exhibit anti-inflammatory, antioxidant, and antiviral effects. However the molecular mechanism underlying these effects has not been well characterized. Because NF-kappaB plays an important role in these processes, it is possible that mangiferin modulates NF-kappaB activation. Our results show that mangiferin blocks tumor necrosis factor (TNF)-induced NF-kappaB activation and NF-kappaB-dependent genes like ICAM1 and COX2. The effect was mediated through inhibition of IKK activation and subsequent blocking of phosphorylation and degradation of IkappaBalpha.In addition, mangiferin inhibits TNF-induced p65 phosphorylation as well as translocation to the nucleus and also inhibits NF-kappaB activation induced by other inflammatory agents like PMA, ceramide, and SA-LPS. Mangiferin, similar to the other known antioxidants, NAC and PDTC, inhibits TNF-induced reactive oxygen intermediate (ROI) generation. Since intracellular glutathione (GSH) levels are known to modulate NF-kappaB levels, we measured the levels of GSH. Mangiferin enhances glutathione level by almost 2-fold more than other anti-oxidants, and at the same time it decreases the levels of GSSG and increases the activity of catalase. Depletion of GSH by buthionine sulfoximine led to a significant reversal of mangiferin effect. Hence mangiferin with its ability to inhibit NF-kappaB and increase the intracellular GSH levels may prove to be a potent drug for anti-inflammatory and antioxidant therapy. Mangiferin-mediated down-regulation of NF-kappaB also potentiates chemotherapeutic agent-mediated cell death, suggesting a role in combination therapy for cancer.     

A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization

The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.     

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.     

Activation of Jun N-terminal kinase/stress-activated protein kinase pathway by tumor necrosis factor alpha leads to intercellular adhesion molecule-1 expression.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine implicated in the pathogenesis of numerous chronic and acute inflammatory conditions. We have previously shown that mouse Sertoli cells respond to TNF-alpha by increasing interleukin-6 production and intercellular adhesion molecule-1 (ICAM-1) expression (1). In this cell type TNF-alpha activates the mitogen-activated protein kinase (MAPK) pathways p42/p44 MAPK, JNK/SAPK, and p38, the last of which is responsible for interleukin-6 production (1). To determine which MAPK signaling pathway is required for TNF-alpha induction of ICAM-1 expression, we have utilized the protein kinase inhibitor dimethylaminopurine, demonstrating that treatment of Sertoli cells with such compound significantly reduced ICAM-1 expression and JNK/SAPK activation. Moreover, dimethylaminopurine treatment increased the expression of MAPK phosphatase-2, providing a possible mechanism of action of this compound. By using agonist antibodies to p55 and to p75 TNF-alpha receptors and both human and mouse TNF-alpha, we demonstrate that both TNF receptors are expressed and that only the p55 receptor is involved in ICAM-1 expression. The p55 receptor activates all of the three pathways, whereas p75 failed to activate any of the MAPKs. Altogether our results demonstrate that TNF-alpha up-regulates ICAM-1 expression through the activation of the JNK/SAPK transduction pathway mediated by the p55 receptor.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

Phosphorylation of Par-4 by protein kinase A is critical for apoptosis

Despite distinct dissimilarities, diverse cancers express several common protumorigenic traits. We present here evidence that the proapoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated protein kinase A (PKA) activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC (selective for apoptosis induction in cancer cells) domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide-, or small interfering RNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA- and phosphorylated T155-dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a procancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.     

TRAF2 phosphorylation modulates tumor necrosis factor alpha-induced gene expression and cell resistance to apoptosis

TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IkappaB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-alpha) stimulation. Although the downstream events in TNF-alpha signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-alpha and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-kappaB target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-alpha-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-alpha stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-alpha-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-kappaB activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-alpha-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-kappaB activity in certain human cancers.     

Mechanisms of apoptosis by the tumor suppressor Par-4

Par‐4 is a pro‐apoptotic, tumor suppressor protein that induces apoptosis selectively in cancer cells. Endoplasmic reticulum‐stress and higher levels of protein kinase A in tumor cells confer the coveted feature of cancer selective response to extracellular and intracellular Par‐4, respectively. Recent studies have shown that systemic Par‐4 confers resistance to tumor growth in mice, and that tumor‐resistance is transferable by bone‐marrow transplantation. Moreover, recombinant Par‐4 inhibits the growth of tumors in mice. As systemic Par‐4 induces apoptosis via cell surface GRP78, strategies that promote GRP78 trafficking to the cell surface are expected sensitize cancer cells to circulating levels of Par‐4. This review illustrates the domains and mechanisms by which Par‐4 orchestrates the apoptotic process in both cell culture models and in physiological settings. J. Cell. Physiol. 227: 3715–3721, 2012. © 2012 Wiley Periodicals, Inc.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Hypoxia-inducible factor mediates hypoxic and tumor necrosis factor alpha-induced increases in tumor necrosis factor-alpha converting enzyme/ADAM17 expression by synovial cells

Chronic hypoxia and inflammatory cytokines are hallmarks of inflammatory joint diseases like rheumatoid arthritis (RA), suggesting a link between this microenvironment and central pathological events. Because TACE/ADAM17 is the predominant protease catalyzing the release of tumor necrosis factor alpha (TNFalpha), a cytokine that triggers a cascade of events leading to RA, we examined the regulation of this metalloprotease in response to hypoxia and TNFalpha itself. We report that low oxygen concentrations and TNFalpha enhance TACE mRNA levels in synovial cells through direct binding of hypoxia-inducible factor-1 (HIF-1) to the 5' promoter region. This is associated with elevated TACE activity as shown by the increase in TNFalpha shedding rate. By the use of HIF-1-deficient cells and by obliterating NF-kappaB activation, it was determined that the hypoxic TACE response is mediated by HIF-1 signaling, whereas the regulation by TNFalpha also requires NF-kappaB activation. As a support for the in vivo relevance of the HIF-1 axis for TACE regulation, immunohistological analysis of TACE and HIF-1 expression in RA synovium indicates that TACE is up-regulated in both fibroblast- and macrophage-like synovial cells where it localizes with elevated expression of both HIF-1 and TNFalpha. These findings suggest a mechanism by which TACE is increased in RA-affected joints. They also provide novel mechanistic clues on the influence of the hypoxic and inflammatory microenvironment on joint diseases.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

Reduced expression of adipose triglyceride lipase enhances tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-kappaB

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.     

Activation of nuclear factor kappaB and induction of apoptosis by tumor necrosis factor-alpha in the mouse uterine epithelial WEG-1 cell line

In order to better understand how tumor necrosis factor (TNF)-alpha may contribute to the local regulation of uterine cell death, cultures of mouse uterine epithelial WEG-1 cells were exposed to TNF-alpha and observed at different time intervals. Earliest decrease in cell viability was observed after 31 h of exposure to 50 ng/ml mouse TNF-alpha and was associated with the expression of several markers of apoptosis. Treatment with human TNF-alpha or addition of a neutralizing antibody against TNF-alpha receptor protein 80 to mouse TNF-alpha resulted in attenuated induction of apoptosis, suggesting that coengagement of the two TNF-alpha receptor types is required for maximal impact. Ceramide analogs failed to replicate the effect of TNF-alpha and the stress-activated protein kinase signaling pathway was not activated by the cytokine. Treatment with mouse TNF-alpha resulted in an increase in nuclear factor (NF)kappaB activity that receded after 24 h. The impact of human TNF-alpha on NFkappaB activation was more moderate. Addition of either one of three different inhibitors of NFkappaB (SN50, PDTC, and A771726) to mouse TNF-alpha sensitized WEG-1 cells to the toxicity of the cytokine. Our data suggest that WEG-1 cells initiate their response to TNF-alpha with an increase in NFkappaB activation that may have transiently biased these cells toward cell death resistance.