Production of nitric oxide and tumor necrosis factor-alpha by Smilacis rhizoma in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which, Smilacis rhizoma (SR) regulates nitric oxide (NO) production. When SR was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production. However, SR had no effect on NO production by itself. The increased production of NO from rIFN-gamma plus SR-stimulated cells was almost completely inhibited by pre-treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa B (NF-kappaB). Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus SR caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of SR on TNF-alpha production significantly. These findings demonstrate that SR increases the production of NO and TNF-alpha by rIFN-gamma-primed macrophages and suggest that NF-kappaB plays a critical role in mediating these effects of SR.     

Decreased epithelial barrier function evoked by exposure to metabolic stress and nonpathogenic E. coli is enhanced by TNF-alpha

A defect in mitochondrial activity contributes to many diseases. We have shown that monolayers of the human colonic T84 epithelial cell line exposed to dinitrophenol (DNP, uncouples oxidative phosphorylation) and nonpathogenic Escherichia coli (E. coli) (strain HB101) display decreased barrier function. Here the impact of DNP on macrophage activity and the effect of TNF-alpha, DNP, and E. coli on epithelial permeability were assessed. DNP treatment of the human THP-1 macrophage cell line resulted in reduced ATP synthesis, and, although hyporesponsive to LPS, the metabolically stressed macrophages produced IL-1beta, IL-6, and TNF-alpha. Given the role of TNF-alpha in inflammatory bowel disease (IBD) and the association between increased permeability and IBD, recombinant TNF-alpha (10 ng/ml) was added to the DNP (0.1 mM) + E. coli (10(6) colony-forming units), and this resulted in a significantly greater loss of T84 epithelial barrier function than that elicited by DNP + E. coli. This increased epithelial permeability was not due to epithelial death, and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF-kappabeta signaling (pyrrolidine dithiocarbamate, NF-kappabeta essential modifier-binding peptide, BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in transepithelial electrical resistance was unaffected by the inhibitors of NF-kappabeta. Thus, as an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and, as such, could contribute to existing inflammation or trigger relapses in IBD. Thus metabolically stressed epithelia display increased permeability in the presence of viable nonpathogenic E. coli that is exaggerated by TNF-alpha released by activated immune cells, such as macrophages, that retain this ability even if they themselves are experiencing a degree of metabolic stress.     

TNF-alpha-induced cyclooxygenase-2 expression in human lung epithelial cells: involvement of the phospholipase C-gamma 2, protein kinase C-alpha, tyrosine kinase, NF-kappa B-inducing kinase, and I-kappa B kinase 1/2 pathway

TNF-alpha induced a dose- and time-dependent increase in cyclooxygenase-2 (COX-2) expression and PGE2 formation in human NCI-H292 epithelial cells. Immunofluorescence staining demonstrated that COX-2 was expressed in cytosol and nuclear envelope. Tyrosine kinase inhibitors (genistein or herbimycin) or phosphoinositide-specific phospholipase C inhibitor (U73122) blocked TNF-alpha-induced COX-2 expression. TNF-alpha also stimulated phosphatidylinositol hydrolysis and protein kinase C (PKC) activity, and both were abolished by genistein or U73122. The PKC inhibitor, staurosporine, also inhibited TNF-alpha-induced response. The 12-O-tetradecanoylphorbol 13-acetate (TPA), a PKC activator, also stimulated COX-2 expression, this effect being inhibited by genistein or herbimycin. NF-kappaB DNA-protein binding and COX-2 promoter activity were enhanced by TNF-alpha, and these effects were inhibited by genistein, U73122, staurosporine, or pyrolidine dithiocarbamate. TPA stimulated both NF-kappaB DNA-protein binding and COX-2 promoter activity, these effects being inhibited by genistein, herbimycin, or pyrolidine dithiocarbamate. The TNF-alpha-induced, but not the TPA-induced, COX-2 promoter activity was inhibited by phospholipase C-gamma2 mutants, and the COX-2 promoter activity induced by either agent was attenuated by dominant-negative mutants of PKC-alpha, NF-kappaB-inducing kinase, or I-kappaB (inhibitory protein that dissociates from NF-kappaB) kinase (IKK)1 or 2. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by staurosporine or herbimycin. These results suggest that, in NCI-H292 epithelial cells, TNF-alpha might activate phospholipase C-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and protein tyrosine kinase, resulting in the activation of NF-kappaB-inducing kinase and IKK1/2, and NF-kappaB in the COX-2 promoter, then initiation of COX-2 expression and PGE2 release.     

The TRAIL apoptotic pathway mediates proteasome inhibitor induced apoptosis in primary chronic lymphocytic leukemia cells

The proteasome inhibitors are a new class of antitumor agents. These inhibitors cause the accumulation of many proteins in the cell with the induction of apoptosis including TRAIL death receptors DR4 and DR5, but the role of the TRAIL apoptotic pathway in proteasome inhibitor cytotoxicity is unknown. Herein, we have demonstrated that the induction of apoptosis by the proteasome inhibitors, MG-132 and PS-341 (bortezomib, Velcade), in primary CLL cells and the Burkitt lymphoma cell line, BJAB, is associated with up-regulation of TRAIL and its death receptors, DR4 and DR5. In addition, FLICE-like inhibitory protein (c-FLIP) protein is decreased. MG-132 treatment increases binding of DR5 to the adaptor protein FADD, and causes caspase-8 activation and cleavage of pro-apoptotic BID. Moreover, DR4:Fc or blockage of DR4 and DR5 expression using RNA interference, which prevents TRAIL apoptotic signaling, blocks proteasome inhibitor induced apoptosis. MG-132 also increases apoptosis and DR5 expression in normal B-cells. However, when the proteasome inhibitors are combined with TRAIL or TRAIL receptor activating antibodies the amount of apoptosis is increased in CLL cells but not in normal B cells. Thus, activation of the TRAIL apoptotic pathway contributes to proteasome inhibitor induced apoptosis in CLL cells.     

Resveratrol attenuates TNF-alpha-induced activation of coronary arterial endothelial cells: role of NF-kappaB inhibition

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its cardioprotective effects are not completely understood. Because TNF-alpha-induced endothelial activation and vascular inflammation play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits TNF-alpha-induced signal transduction in human coronary arterial endothelial cells (HCAECs). We found that TNF-alpha significantly increased adhesiveness of the monocytic THP-1 cells to HCAECs, an effect that could be inhibited by pretreatment with resveratrol and the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Previously, we found that TNF-alpha activates NAD(P)H oxidases, and our recent data showed that TNF-alpha-induced endothelial activation was prevented by the NAD(P)H oxidase inhibitor apocynin or catalase plus SOD. Resveratrol also inhibited H(2)O(2)-induced monocyte adhesiveness. Using a reporter gene assay, we found that, in HCAECs, TNF-alpha significantly increased NF-kappaB activity, which could be inhibited by resveratrol (>50% inhibition at 10(-6) mol/l) and pyrrolidine dithiocarbamate. Resveratrol also inhibited TNF-alpha-induced, NF-kappaB-driven luciferase expression in rat aortas electroporated with the reporter gene construct. In TNF-alpha-treated HCAECs, resveratrol (in the submicromolar range) significantly attenuated expression of NF-kappaB-dependent inflammatory markers inducible nitric oxide synthase, IL-6, bone morphogenetic protein-2, ICAM-1, and VCAM. Thus resveratrol at nutritionally relevant concentrations inhibits TNF-alpha-induced NF-kappaB activation and inflammatory gene expression and attenuates monocyte adhesiveness to HCAECs. We propose that these anti-inflammatory actions of resveratrol are responsible, at least in part, for its cardioprotective effects.     

The Proteasome Inhibitor MG-132 Induces AIF Nuclear Translocation Through Down-Regulation of ERK and Akt/mTOR Pathway

Anticancer activity of proteasome inhibitors has been demonstrated in various cancer cell types. However, mechanisms by which they exert anticancer action were not fully understood. The present study was undertaken to examine the effect of the proteasome inhibitor MG-132 and the underlying mechanism in glioma cells. MG-132 caused alterations in mitochondrial membrane potential and apoptosis-inducing factor (AIF) nuclear translocation. MG-132 induced reduction in ERK and Akt activation. The transient transfection of constitutively active forms of MEK, an upstream of ERK, and Akt blocked the MG-132-induced cell death. Similarly to down-regulation of Akt, expression levels of mTOR were inhibited by MG-132. Addition of rapamycin, an inhibitor of mTOR, caused stimulation of the MG-132-induced cell death. There were no significant changes in levels of XIAP, survivin, and Bax. Overexpression of constitutively active forms of MEK and Akt blocked the MG-132-induced AIF nuclear translocation. These findings indicate that MG-132 induces AIF nuclear translocation through down-regulation of ERK and Akt/mTOR pathways. These data suggest that proteasome inhibitors may serve as potential therapeutic agents for malignant human gliomas.     

NF-kappaB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells

The maintenance of intestinal mucosal integrity depends on a balance between cell renewal and cell death, including apoptosis. The natural polyamines, putrescine, spermidine, and spermine, are essential for mucosal growth, and decreasing polyamine levels cause G(1) phase growth arrest in intestinal epithelial (IEC-6) cells. The present study was done to determine changes in susceptibility of IEC-6 cells to apoptosis after depletion of cellular polyamines and to further elucidate the role of nuclear factor-kappaB (NF-kappaB) in this process. Although depletion of polyamines by alpha-difluoromethylornithine (DFMO) did not directly induce apoptosis, the susceptibility of polyamine-deficient cells to staurosporine (STS)-induced apoptosis increased significantly as measured by changes in morphological features and internucleosomal DNA fragmentation. In contrast, polyamine depletion by DFMO promoted resistance to apoptotic cell death induced by the combination of tumor necrosis factor-alpha (TNF-alpha) and cycloheximide. Depletion of cellular polyamines also increased the basal level of NF-kappaB proteins, induced NF-kappaB nuclear translocation, and activated the sequence-specific DNA binding activity. Inhibition of NF-kappaB binding activity by sulfasalazine or MG-132 not only prevented the increased susceptibility to STS-induced apoptosis but also blocked the resistance to cell death induced by TNF-alpha in combination with cycloheximide in polyamine-deficient cells. These results indicate that 1) polyamine depletion sensitizes intestinal epithelial cells to STS-induced apoptosis but promotes the resistance to TNF-alpha-induced cell death, 2) polyamine depletion induces NF-kappaB activation, and 3) disruption of NF-kappaB function is associated with altered susceptibility to apoptosis induced by STS or TNF-alpha. These findings suggest that increased NF-kappaB activity after polyamine depletion has a proapoptotic or antiapoptotic effect on intestinal epithelial cells determined by the nature of the death stimulus.     

Dual activity of pyrrolidine dithiocarbamate on kappaB-dependent gene expression in U937 cells: II. Regulation by tumour necrosis factor-alpha.

In the human promonocytic U937 cell line, pyrrolidine dithiocarbamate (PDTC) was a potent inhibitor of the nuclear factor-kappaB (NF-kappaB) signalling pathway induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). However, PDTC did not inhibit tumour necrosis factor-alpha (TNF-alpha)-induced NF-kappaB DNA binding activity but potentiated the effect of TNF-alpha on kappaB-dependent gene expression. The stimulatory effect of PDTC with TNF-alpha was not observed with an HIV-1 LTR reporter construct containing two mutated kappaB binding sites or with a construct with a mutation of the activating protein (AP)-2 binding site located between the two kappaB elements. Two distinct signalling pathways, one mediated by TPA and the other by TNF-alpha, were shown to interact, functionally defining a threshold important in the inhibitory or stimulatory effect of PDTC on kappaB-dependent gene expression. Evidence that PDTC induced AP-1 DNA binding and AP-1 reporter gene activity, raised the hypothesis that the effect of PDTC was mediated by an interaction between the AP-1 pathway and p65(RelA). Co-transfection with expression vectors for p65(RelA) and the AP-1 subunits c-Fos and c-Jun resulted in a decrease in the stimulatory effect of PDTC on HIV-1 LTR activity. Co-transfection of p65(RelA) with Tam67, a dominant negative mutant of c-Jun defective in transactivation, stimulated the effect of PDTC on HIV-1 LTR activity. Evidence that the stimulatory effect of Tam67 with PDTC was reduced with c-Jun is consistent with the hypothesis.     

p38 MAPK and NF-kappaB mediate COX-2 expression in human airway myocytes

We have previously demonstrated that p38 and extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinases (MAPK) are components of proinflammatory induced cytokine expression in human airway myocytes. The experiments described here further these studies by examining p38 MAPK and NF-kappaB regulation of cyclooxygenase-2 (COX-2) expression in response to a complex inflammatory stimulus consisting of 10 ng/ml interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and interferon (IFN)-gamma. COX-2 expression was induced with this stimulus in a time-dependent manner, with maximal expression seen 12-20 h after treatment. Semiquantitative RT-PCR and immunoblotting experiments demonstrate decreased COX-2 expression following treatment with the p38 MAPK inhibitor SB-203580 (25 microM) or the proteosome inhibitor MG-132 (1 microM). SB-203580 did not affect cytokine-stimulated IkappaBalpha degradation, NF-kappaB nuclear binding activity, or NF-kappaB-dependent signaling from the COX-2 promoter, indicating that p38 MAPK and NF-kappaB may affect COX-2 expression via separate signaling pathways. SB-203580, but not MG-132, also increased the initial rate of COX-2 mRNA decay, indicating p38 MAPK, but not NF-kappaB, participates in the regulation of COX-2 mRNA stability. These findings suggest that although p38 MAPK and NF-kappaB signaling regulate steady-state levels of COX-2 expression, p38 MAPK additionally affects stability of COX-2 mRNA in cytokine-stimulated human airway myocytes.     

Leonurus sibiricus induces nitric oxide and tumor necrosis factor-alpha in mouse peritoneal macrophages

Using mouse peritoneal macrophages, we have examined the mechanism by which Leonurus sibiricus (LS) regulates nitric oxide (NO) production. When LS was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO production; however, LS by itself had no effect on NO production. The increased production of NO from rIFN-gamma plus LS-stimulated cells was almost completely inhibited by pretreatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappaB. Furthermore, treatment of peritoneal macrophages with rIFN-gamma plus LS caused a significant increase in tumor necrosis factor-alpha (TNF-alpha) production. PDTC also decreased the effect of LS on TNF-alpha production significantly. Because NO and TNF-alpha play an important role in immune function and host defense, LS treatment could modulate several aspects of host defense mechanisms as a result of stimulation of the inducible nitric oxide synthase.     

Enac degradation in A6 cells by the ubiquitin-proteosome proteolytic pathway

Amiloride-sensitive epithelial Na(+) channels (ENaC) are responsible for trans-epithelial Na(+) transport in the kidney, lung, and colon. The channel consists of three subunits (alpha, beta, gamma) each containing a proline rich region (PPXY) in their carboxyl-terminal end. Mutations in this PPXY domain cause Liddle's syndrome, an autosomal dominant, salt-sensitive hypertension, by preventing the channel's interactions with the ubiquitin ligase Neural precursor cell-expressed developmentally down-regulated protein (Nedd4). It is postulated that this results in defective endocytosis and lysosomal degradation of ENaC leading to an increase in ENaC activity. To show the pathway that degrades ENaC in epithelial cells that express functioning ENaC channels, we used inhibitors of the proteosome and measured sodium channel activity. We found that the inhibitor, MG-132, increases amiloride-sensitive trans-epithelial current in Xenopus distal nephron A6 cells. There also is an increase of total cellular as well as membrane-associated ENaC subunit molecules by Western blotting. MG-132-treated cells also have increased channel density in patch clamp experiments. Inhibitors of lysosomal function did not reproduce these findings. Our results suggest that in native renal cells the proteosomal pathway is an important regulator of ENaC function.