Sulindac inhibits activation of the NF-kappaB pathway.

Sulindac is a non-steroidal anti-inflammatory agent that is related both structurally and pharmacologically to indomethacin. In addition to its anti-inflammatory properties, sulindac has been demonstrated to have a role in the prevention of colon cancer. Both its growth inhibitory and anti-inflammatory properties are due at least in part to its ability to decrease prostaglandin synthesis by inhibiting the activity of cyclooxygenases. Recently, we demonstrated that both aspirin and sodium salicylate, but not indomethacin, inhibited the activity of an IkappaB kinase beta (IKKbeta) that is required to activate the nuclear factor-kappaB (NF-kappaB) pathway. In this study, we show that sulindac and its metabolites sulindac sulfide and sulindac sulfone can also inhibit the NF-kappaB pathway in both colon cancer and other cell lines. Similar to our previous results with aspirin, this inhibition is due to sulindac-mediated decreases in IKKbeta kinase activity. Concentrations of sulindac that inhibit IKKbeta activity also reduce the proliferation of colon cancer cells. These results suggest that the growth inhibitory and anti-inflammatory properties of sulindac may be regulated in part by inhibition of kinases that regulate the NF-kappaB pathway.     

Impairment of NF-kappaB activation and modulation of gene expression by calpastatin

To address the involvement of the calpain systemin both basal and silica-induced nuclear factor (NF)-B activation,several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IB)degradation and NF-B activation were observed in cells stablyoverexpressing calpastatin. In addition, the cells in which calpain wasconstitutively inhibited by the overexpression of calpastatin exhibiteda notable morphological change. Whereas empty vector-transfected cellsdisplayed a morphology indistinguishable from that of parental cells,cells overexpressing calpastatin exhibited a mosaic morphologicalchange with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genescan be evaluated for their expression at the same time, showed thatcalpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen 2,DAZ, and mitochondrial capsule selenoprotein. These results suggestthat, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.

     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway

4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells.     

ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation

Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.     

Suppression of MEK/ERK signaling pathway enhances cisplatin-induced NF-kappaB activation by protein phosphatase 4-mediated NF-kappaB p65 Thr dephosphorylation

We previously reported that suppression of the MEK/ERK pathway increases drug resistance of SiHa cells. In this study, we further characterized the underlying mechanism of this phenomenon. Pretreatment of SiHa cells with MEK/ERK inhibitor enhanced cisplatin-induced NF-kappaB activation. However, results of immunoblotting analysis showed that neither cisplatin nor MEK/ERK inhibitors induced marked IkappaBalpha degradation, suggesting that suppression of the MEK/ERK signaling pathway may enhance cisplatin-induced NF-kappaB activation via mechanisms other than the conventional pathway. Previous findings that protein phosphatase 4 (PP4), a nuclear serine/threonine phosphatase, directly interacts with and activates NF-kappaB led us to examine the phosphorylation status of NF-kappaB p65. Coincident with activation of NF-kappaB, cisplatin induced Ser phosphorylation but decreased Thr phosphorylation of NF-kappaB p65. Suppression of the MEK/ERK pathway further enhanced cisplatin-induced Thr dephosphorylation but did not affect cisplatin-induced Ser phosphorylation of NF-kappaB p65. Further, in parallel with Thr dephosphorylation, the protein level of nuclear PP4 was increased in cisplatin-treated cells and was further increased by suppression of the MEK/ERK pathway. SiHa cells were then transfected by a sense or an antisense PP4 gene. PP4-overexpressing cells showed a decrease in Thr phosphorylation of NF-kappaB p65 to nearly undetectable levels, and both basal and cisplatin-induced NF-kappaB activities were higher than those in parental cells. By contrast, cisplatin, either alone or with MEK/ERK inhibitors, induced little NF-kappaB activation in antisense PP4-transfected cells. Coprecipitated complex kinase assay revealed a fragment of NF-kappaB p65 (amino acids 279-444) to contain potential phosphorylation sites that directly interact with PP4. Further studies by site-directed mutagenesis suggested that Thr(435) was the major phosphorylation site.     

Anticancer cardamonin analogs suppress the activation of NF-kappaB pathway in lung cancer cells

NF-kappaB pathway has been proven to be critical to survival of lung cancer cells, and many natural products from plants were shown to inhibit the activation of this pathway. In this study, we investigated the effects of two cardamonin analogs, 4,4′-dihydroxylchalcone (DHC) and 4,4′-dihydroxy-2′-methoxychalcone (DHMC), on survival of lung cancer cells and the involved mechanisms. MTT assay revealed that the two compounds potently decreased the survival of immortalized and primary lung cancer cells. DHC and DHMC were able to induce apoptosis in A549 and NCI-H460 cells. Immunoblotting, immunofluorescent staining, and luciferase reporter further demonstrated that the two compounds suppressed the activation of NF-kappaB pathway in lung cancer cells. PMA-mediated NF-kappaB reactivation abrogated the effect of DHC and DHMC on lung cancer cells. DHC and DHMC were also shown to suppress the growth of A549 xenograft in mice. Collectively, we verified two cardamonin analogs as novel compounds suppressing NF-kappaB signaling for lung cancer therapy.     

Fer and Fps/Fes participate in a Lyn-dependent pathway from FcepsilonRI to platelet-endothelial cell adhesion molecule 1 to limit mast cell activation

Mast cells express the high affinity IgE receptor FcepsilonRI, which upon aggregation by multivalent antigens elicits signals that cause rapid changes within the mast cell and in the surrounding tissue. We previously showed that FcepsilonRI aggregation caused a rapid increase in phosphorylation of both Fer and Fps/Fes kinases in bone marrow-derived mast cells. In this study, we report that FcepsilonRI aggregation leads to increased Fer/Fps kinase activities and that Fer phosphorylation downstream of FcepsilonRI is independent of Syk, Fyn, and Gab2 but requires Lyn. Activated Fer/Fps readily phosphorylate the C terminus of platelet-endothelial cell adhesion molecule 1 (Pecam-1) on immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a non-ITIM residue (Tyr(700)) in vitro and in transfected cells. Mast cells devoid of Fer/Fps kinase activities display a reduction in FcepsilonRI aggregation-induced tyrosine phosphorylation of Pecam-1, with no defects in recruitment of Shp1/Shp2 phosphatases observed. Lyn-deficient mast cells display a dramatic reduction in Pecam-1 phosphorylation at Tyr(685) and a complete loss of Shp2 recruitment, suggesting a role as an initiator kinase for Pecam-1. Consistent with previous studies of Pecam-1-deficient mast cells, we observe an exaggerated degranulation response in mast cells lacking Fer/Fps kinases at low antigen dosages. Thus, Lyn and Fer/Fps kinases cooperate to phosphorylate Pecam-1 and activate Shp1/Shp2 phosphatases that function in part to limit mast cell activation.     

Polyubiquitination events mediate polymethylmethacrylate (PMMA) particle activation of NF-kappaB pathway

The pathologic response to implant wear-debris constitutes a major component of inflammatory osteolysis and remains under intense investigation. Polymethylmethacrylate (PMMA) particles, which are released during implant wear and loosening, constitute a major culprit by virtue of inducing inflammatory and osteolytic responses by macrophages and osteoclasts, respectively. Recent work by several groups has identified important cellular entities and secreted factors that contribute to inflammatory osteolysis. In previous work, we have shown that PMMA particles contribute to inflammatory osteolysis through stimulation of major pathways in monocytes/macrophages, primarily NF-κB and MAP kinases. The former pathway requires assembly of large IKK complex encompassing IKK1, IKK2, and IKKγ/NEMO. We have shown recently that interfering with the NF-κB and MAPK activation pathways, through introduction of inhibitors and decoy molecules, impedes PMMA-induced inflammation and osteolysis in mouse models of experimental calvarial osteolysis and inflammatory arthritis. In this study, we report that PMMA particles activate the upstream transforming growth factor β-activated kinase-1 (TAK1), which is a key regulator of signal transduction cascades leading to activation of NF-κB and AP-1 factors. More importantly, we found that PMMA particles induce TAK1 binding to NEMO and UBC13. In addition, we show that PMMA particles induce TRAF6 and UBC13 binding to NEMO and that lack of TRAF6 significantly attenuates NEMO ubiquitination. Altogether, these observations suggest that PMMA particles induce ubiquitination of NEMO, an event likely mediated by TRAF6, TAK1, and UBC13. Our findings provide important information for better understanding of the mechanisms underlying PMMA particle-induced inflammatory responses.     

LIND/ABIN-3 is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation

Recognition of lipopolysaccharide (LPS) by Toll-like receptor (TLR)4 initiates an intracellular signaling pathway leading to the activation of nuclear factor-kappaB (NF-kappaB). Although LPS-induced activation of NF-kappaB is critical to the induction of an efficient immune response, excessive or prolonged signaling from TLR4 can be harmful to the host. Therefore, the NF-kappaB signal transduction pathway demands tight regulation. In the present study, we describe the human protein Listeria INDuced (LIND) as a novel A20-binding inhibitor of NF-kappaB activation (ABIN) that is related to ABIN-1 and -2 and, therefore, is further referred to as ABIN-3. Similar to the other ABINs, ABIN-3 binds to A20 and inhibits NF-kappaB activation induced by tumor necrosis factor, interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate. However, unlike the other ABINs, constitutive expression of ABIN-3 could not be detected in different human cells. Treatment of human monocytic cells with LPS strongly induced ABIN-3 mRNA and protein expression, suggesting a role for ABIN-3 in the LPS/TLR4 pathway. Indeed, ABIN-3 overexpression was found to inhibit NF-kappaB-dependent gene expression in response to LPS/TLR4 at a level downstream of TRAF6 and upstream of IKKbeta. NF-kappaB inhibition was mediated by the ABIN-homology domain 2 and was independent of A20 binding. Moreover, in vivo adenoviral gene transfer of ABIN-3 in mice reduced LPS-induced NF-kappaB activity in the liver, thereby partially protecting mice against LPS/D-(+)-galactosamine-induced mortality. Taken together, these results implicate ABIN-3 as a novel negative feedback regulator of LPS-induced NF-kappaB activation.