Interferon gamma (IFNgamma ) and tumor necrosis factor alpha synergism in ME-180 cervical cancer cell apoptosis and necrosis. IFNgamma inhibits cytoprotective NF-kappa B through STAT1/IRF-1 pathways

We investigated the molecular mechanism of the synergism between interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) documented in a variety of biological occasions such as tumor cell death and inflammatory responses. IFNgamma/TNFalpha synergistically induced apoptosis of ME-180 cervical cancer cells. IFNgamma induced STAT1 phosphorylation and interferon regulatory factor 1 (IRF-1) expression. Transfection of phosphorylation-defective STAT1 inhibited IFNgamma/TNFalpha-induced apoptosis, whereas IRF-1 transfection induced susceptibility to TNFalpha. Dominant-negative IkappaBalpha transfection sensitized ME-180 cells to TNFalpha. IFNgamma pretreatment attenuated TNFalpha- or p65-induced NF-kappaB reporter activity, whereas it did not inhibit p65 translocation or DNA binding of NF-kappaB. IRF-1 transfection alone inhibited TNFalpha-induced NF-kappaB activity, which was reversed by coactivator p300 overexpression. Caspases were activated by IFNgamma/TNFalpha combination; however, caspase inhibition did not abrogate IFNgamma/TNFalpha-induced cell death. Instead, caspase inhibitors directed IFNgamma/TNFalpha-treated ME-180 cells to undergo necrosis, as demonstrated by Hoechst 33258/propidium iodide staining and electron microscopy. Taken together, our results indicate that IFNgamma and TNFalpha synergistically act to destroy ME-180 tumor cells by either apoptosis or necrosis, depending on caspase activation, and STAT1/IRF-1 pathways initiated by IFNgamma play a critical role in IFNgamma/TNFalpha synergism by inhibiting cytoprotective NF-kappaB. IFNgamma/TNFalpha synergism appears to activate cell death machinery independently of caspase activation, and caspase activation seems to merely determine the mode of cell death.     

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.     

Inactivation of the inhibitory kappaB protein kinase/nuclear factor kappaB pathway by Par-4 expression potentiates tumor necrosis factor alpha-induced apoptosis.

Par-4 is a novel protein identified in cells undergoing apoptosis. The ability of Par-4 to promote apoptotic cell death is dependent on the binding and inactivation of the atypical protein kinases C (PKCs). This subfamily of kinases has been reported to control nuclear factor kappaB (NF-kappaB) through the regulation of the IkappaB kinase activity. NF-kappaB activation by tumor necrosis factor alpha (TNFalpha) provides a survival signal that impairs the TNFalpha-induced apoptotic response. We show here that expression of Par-4 inhibits the TNFalpha-induced nuclear translocation of p65 as well as the kappaB-dependent promoter activity. Interestingly, Par-4 expression blocks inhibitory kappaB protein (IkappaB) kinase activity, which leads to the inhibition of IkappaB phosphorylation and degradation, in a manner that is dependent on its ability to inhibit lambda/iotaPKC. Of potential functional relevance, the expression of Par-4 allows TNFalpha to induce apoptosis in NIH-3T3 cells. In addition, the down-regulation of Par-4 levels by oncogenic Ras sensitizes cells to TNFalpha-induced NF-kappaB activation.     

Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha -induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells.

In this study, we explored a novel function of polymorphonuclear neutrophils (PMN) NAD(P)H oxidase in the mechanism of tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells. Studies were made in mice lacking the p47(phox) subunit of NAD(P)H oxidase as well as in cultured mouse lung vascular endothelial cells (MLVEC) from these mice. In response to TNFalpha challenge, NF-kappaB activation and ICAM-1 expression were significantly attenuated in lungs of p47(phox)(-/-) mice as compared with wild-type (WT) mice. The attenuated NF-kappaB activation in p47(phox)(-/-) mice was secondary to inhibition of NIK activity and subsequent IkappaBalpha degradation. Induction of neutropenia using anti-PMN serum prevented the initial TNFalpha-induced NF-kappaB activation and ICAM-1 expression in WT mice, indicating the involvement of PMN NAD(P)H oxidase in signaling these responses. Moreover, the responses were restored upon repletion with PMN obtained from WT mice but not with PMN from p47(phox)(-/-) mice. These findings were recapitulated in MLVEC co-cultured with PMN, suggesting that NF-kappaB activation and resultant ICAM-1 expression in endothelial cells occurred secondary to oxidants generated by the PMN NAD(P)H oxidase complex. The functional relevance of the PMN NAD(P)H oxidase in mediating TNFalpha-induced ICAM-1-dependent endothelial adhesivity was evident by markedly reduced adhesion of p47(phox)(-/-) PMN in co-culture experiments. Thus, oxidant signaling by the PMN NAD(P)H oxidase complex is an important determinant of TNFalpha-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.     

Hypoxia inducing factor-1α regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia

Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1α is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1α (HIF-1α) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1α protein. Hypoxia- and DEF-induced HIF-1α activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1α inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1α using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1α inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1α inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells.     

Inhibition of phosphoinositide 3 kinase-Akt (protein kinase B)-nuclear factor-kappa B pathway by lovastatin limits endothelial-monocyte cell interaction

Integrity of the blood-brain barrier is essential for the normal functioning of CNS. Its disruption contributes to the pathobiology of various inflammatory neurodegenerative disorders. We have shown that the HMG-CoA reductase inhibitor (lovastatin) attenuated experimental autoimmune encephalomyelitis (EAE, an inflammatory disease of CNS) in rodents by inhibiting the infiltration of mononuclear cells into the CNS. Here, using an in vitro system, we report that lovastatin inhibits endothelial-monocyte cell interaction by down-regulating the expression of vascular cell adhesion molecule-1 and E-selectin by inhibiting the phosphoinositide 3 kinase (PI3-kinase)/protein kinase B (Akt)/nuclear factor-kappa B (NF-kappaB) pathway in endothelial cells. It inhibits tumor necrosis factor alpha (TNFalpha)-induced PI3-kinase, Akt and NF-kappaB activation in these cells. Co-transfection of constitutively active forms of PI3-kinase and Akt reversed the lovastatin-mediated inhibition of TNFalpha-induced adhesion, as well as activation of NF-kappaB, indicating the involvement of the PI3-kinase/Akt pathway in the interaction of adhesion molecules and the process of adhesion. This study reports that lovastatin down-regulates the pathway affecting the expression and interaction of adhesion molecules on endothelial cells, which in turn restricts the migration and infiltration of mononuclear cells thereby attenuating the pathogenesis of inflammatory diseases.     

Simvastatin modulates TNFalpha-induced adhesion molecules expression in human endothelial cells

Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDL-cholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor alpha (TNFalpha). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFalpha-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFalpha-induced activation of nuclear factor kappaB (NF-kappaB) and enhanced expression of peroxisome proliferator-activated receptor alpha (PPARalpha). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-kappaB.     

A protective role for the human SMG-1 kinase against tumor necrosis factor-alpha-induced apoptosis

The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.     

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.