Phosphorylation of extracellular signal‐regulated kinase 1/2, p38 mitogen‐activated protein kinase and nuclear translocation of nuclear factor‐κB are involved in upregulation of matrix metalloproteinase‐9 by tumour necrosis factor‐α

Background: Upregulation of matrix metalloproteinase‐9 (MMP‐9) induced by tumour necrosis factor‐α (TNF‐α) is reportedly involved in a variety of non‐neoplastic and neoplastic diseases. In this study, we examined which signalling pathways are involved in TNF‐α‐induced MMP‐9 upregulation in cholangiocarcinoma (CC). Methods: We used two CC cell lines: HuCCT‐1 and CCKS‐1. Results: In an ex vivo study using HuCCT‐1 and CCKS‐1 cells, TNF‐α treatment induced MMP‐9 production and activation via interaction with TNF receptor‐1 (TNF‐R1) but not with TNF receptor‐2 (TNF‐R2), shown by zymography, and increased MMP‐9 promoter activity (luciferase assay). As for the signalling pathway, TNF‐α stimulation led to the phosphorylation of extracellular signal‐regulated kinase 1/2 (Erk1/2) and p38 mitogen‐activated protein kinase (p38MAPK) and translocation of nuclear factor κB (NF‐κB) (p65) into the nuclei. Inhibition studies using SB203580 (inhibitor of p38MAPK), U0126 (inhibitor of mitogen‐activated or extracellular signal‐regulated protein kinase 1/2) and MG132 (inhibitor of NF‐κB) showed that the phosphorylation of Erk1/2 and p38MAPK with activation of NF‐κB was closely related to MMP‐9 upregulation in both cell lines. Conclusion: These data suggest that TNF‐α/TNF‐R1 interaction leads to the phosphorylation of Erk1/2 and p38MAPK and nuclear translocation of NF‐κB, which is closely associated with the production and activation of MMP‐9 in cultured CC cells of HuCTT‐1 and CCKS‐1. Upregulation of MMP‐9 with NF‐κB activation may be involved in the tumour invasion of CC.     

Melatonin potentiates the antiproliferative and pro‐apoptotic effects of ursolic acid in colon cancer cells by modulating multiple signaling pathways

Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is largely distributed in medical herbs and edible plants. Melatonin is an indoleamine compound produced in the pineal gland and also a plant‐derived product. Both UA and melatonin have been shown to inhibit cancer cell growth in numerous studies, but they have never been combined altogether as an anticolon cancer treatment. In this study, we investigated whether the association between UA and melatonin leads to an enhanced antiproliferative and pro‐apoptotic activities in colon cancer SW480 and LoVo cells. We found that combined treatment with UA and melatonin significantly enhanced inhibition of cell viability and migration, promoted changes in cell morphology and spreading, and increased induction of apoptosis, thereby potentiating the effects of UA alone in colon cancer cells. Moreover, we found that the enhanced effects of UA and melatonin combination are mediated through simultaneous modulation of cytochrome c/caspase, MMP9/COX‐2, and p300/NF‐κB signaling pathways. Combined treatment with UA and melatonin triggered the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, induced cleavage of caspase and PARP proteins, enhanced inhibition of MMP9 and COX‐2 expression, promoted p300 and NF‐κB translocation from cell nuclei to cytoplasm, and abrogated NF‐κB binding and p300 recruitment to COX‐2 promoter in colon cancer cells. These results, therefore, demonstrated that melatonin potentiated the antiproliferative and pro‐apoptotic effects of UA in colon cancer cells by modulating multiple signaling pathways and suggest that such a combinational treatment might potentially become an effective way in colon cancer therapy.     

Bradykinin potentiates cytokine‐induced prostaglandin biosynthesis in osteoblasts by enhanced expression of cyclooxygenase 2, resulting in increased RANKL expression

Objective

Bradykinin (BK) stimulates bone resorption in vitro and synergistically potentiates interleukin‐1 (IL‐1)–induced bone resorption and prostaglandin (PG) formation, suggesting that kinins are important in inflammation‐induced bone loss. The present study was undertaken to study 1) the role of the kinin B1 and B2 receptors in the synergistic interaction with IL‐1 and tumor necrosis factor α (TNFα), 2) the molecular mechanisms involved in synergistic enhancement of PG formation, and 3) the effects of kinins on cytokine‐induced expression of RANKL, RANK, and osteoprotegerin (OPG) (the latter being crucial molecules in osteoclast differentiation).

Methods

Formation of PGs, expression of enzymes involved in arachidonic acid metabolism, and expression of RANKL, RANK, and OPG were assessed in the human osteoblastic cell line MG‐63 and in mouse calvarial bones. The role of NF‐κB and MAP kinases was studied using pharmacologic inhibitors.

Results

PGE₂ formation and cyclooxygenase 2 (COX‐2) protein expression were induced by IL‐1β and potentiated by kinins with affinity for the B1 or B2 receptors, resulting in PGE₂‐dependent enhancement of RANKL. The enhancements of PGE₂ formation and COX‐2 were markedly decreased by inhibition of p38 and JNK MAP kinases, whereas inhibition of NF‐κB resulted in abolishment of the PGE₂ response with only slight inhibition of COX‐2.

Conclusion

Kinin B1 and B2 receptors synergistically potentiate IL‐1– and TNFα‐induced PG biosynthesis in osteoblasts by a mechanism involving increased levels of COX‐2, resulting in increased RANKL. The synergistic stimulation is dependent on NF‐κB and MAP kinases. These mechanisms might help to explain the enhanced bone resorption associated with inflammatory disorders, including that in rheumatoid arthritis.

     

Microparticles stimulate the synthesis of prostaglandin E2 via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1

Objective

Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype.

Methods

Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real‐time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF‐κB and activator protein 1 (AP‐1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IκB expression vectors.

Results

Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX‐2), microsomal prostaglandin E synthase 1 (mPGES‐1), and prostaglandin E₂ (PGE₂). In contrast, no up‐regulation of COX‐1, mPGES‐2, cytosolic PGES, or phospholipase A₂ was observed. The induction of PGE₂ was blocked by selective inhibition of COX‐2. Microparticles activated NF‐κB, AP‐1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF‐κB, AP‐1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE₂ by synovial fibroblasts.

Conclusion

These results demonstrate that microparticles up‐regulate the production of PGE₂ in synovial fibroblasts by inducing COX‐2 and mPGES‐1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.

     

NF‐κB protects Behçet's disease T cells against CD95‐induced apoptosis up‐regulating antiapoptotic proteins

Objective

To determine whether prolongation of the inflammatory reaction in patients with Behçet's disease (BD) is related to apoptosis resistance and is associated with the up‐regulation of antiapoptotic factors.

Methods

The percentage of cell death was evaluated by flow cytometry in peripheral blood mononuclear cells from 35 patients with BD and 30 healthy volunteers. The expression levels of antiapoptotic factors and NF‐κB regulatory proteins were measured using Western blotting and immunohistochemical analyses. To down‐regulate NF‐κB nuclear translocation, BD T lymphocytes were exposed in vitro to thalidomide and subjected to transfection with NF‐κB small interfering RNA.

Results

Although CD95 is highly expressed in BD T cells, the absence of sensitivity to CD95‐induced apoptosis observed may be attributable to the inhibitory action of antiapoptotic genes. Immunoblot analysis for major antiapoptotic proteins showed considerable up‐regulation of the short form of cellular FLIP (cFLIP) and Bcl‐x_L in BD activated T cells, while levels of Bcl‐2, caspase 3, and caspase 8 in activated T cells from patients with BD were comparable with those in activated T cells from normal donors. Moreover, expression of IKK and IκB was up‐regulated, whereas NF‐κB translocated to the nucleus in BD T cells, suggesting that NF‐κB activation may modulate the expression of antiapoptotic genes. Interestingly, thalidomide and NF‐κB small interfering RNA down‐regulated cFLIP and Bcl‐x_L expression levels and sensitized BD activated T cells to CD95‐induced apoptosis.

Conclusion

Taken together, these results indicate that NF‐κB contributes to the regulation of the apoptosis‐related factors and death receptors leading to apoptosis resistance in BD T cell subsets. Our results suggest that NF‐κB plays a crucial role in the pathogenesis of BD, and that its pharmacologic control could represent a key strategy in modulating specific immune‐mediated disease.

     

Melatonin ameliorates Aβ42‐induced alteration of βAPP‐processing secretases via the melatonin receptor through the Pin1/GSK3β/NF‐κB pathway in SH‐SY5Y cells

Melatonin is involved in the physiological regulation of the β‐amyloid precursor protein (βAPP)‐cleaving secretases which are responsible for generation of the neurotoxic amyloid beta (Aβ) peptide, one of the hallmarks of Alzheimer's disease (AD) pathology. In this study, we aimed to determine the underlying mechanisms of this regulation under pathological conditions. We establish that melatonin prevents Aβ₄₂‐induced downregulation of a disintegrin and metalloproteinase domain‐containing protein 10 (ADAM10) as well as upregulation of β‐site APP‐cleaving enzyme 1 (BACE1) and presenilin 1 (PS1) in SH‐SY5Y cell cultures. We also demonstrate that the intrinsic mechanisms of the observed effects occurred via regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and glycogen synthase kinase (GSK)‐3β as melatonin reversed Aβ₄₂‐induced upregulation and nuclear translocation of NF‐κBp65 as well as activation of GSK3β via its receptor activation. Furthermore, specific blocking of the NF‐κB and GSK3β pathways partially abrogated the Aβ₄₂‐induced reduction in the BACE1 and PS1 levels. In addition, GSK3β blockage affected α‐secretase cleavage and modulated nuclear translocation of NF‐κB. Importantly, our study for the first time shows that peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 (Pin1) is a crucial target of melatonin. The compromised levels and/or genetic variation of Pin1 are associated with age‐dependent tau and Aβ pathologies and neuronal degeneration. Interestingly, melatonin alleviated the Aβ₄₂‐induced reduction of nuclear Pin1 levels and preserved the functional integrity of this isomerase. Our findings illustrate that melatonin attenuates Aβ₄₂‐induced alterations of βAPP‐cleaving secretases possibly via the Pin1/GSK3β/NF‐κB pathway.     

Acute‐phase serum amyloid A stimulation of angiogenesis,leukocyte recruitment,and matrix degradation in rheumatoid arthritis through an NF‐κB–dependent signal transduction pathway

Objective

To examine the role of the acute‐phase protein serum amyloid A (A‐SAA) in regulating cell adhesion molecule expression, leukocyte recruitment, and angiogenesis in rheumatoid arthritis (RA).

Methods

Intercellular adhesion molecule 1 (ICAM‐1), vascular cell adhesion molecule 1 (VCAM‐1), and matrix metalloproteinase 1 (MMP‐1) expression was examined in RA fibroblast‐like synoviocytes (FLS) and human microvascular endothelial cells (HMVECs) using flow cytometry and enzyme‐linked immunosorbent assay techniques. Peripheral blood mononuclear cell (PBMC) adhesion to FLS/HMVECs was determined by flow cytometry. Angiogenesis was examined using a Boyden chemotaxis chamber and Matrigel tubule formation. NF‐κB/IκBα mediation of the effects of A‐SAA was investigated using a specific NF‐κB inhibitor and Western blotting.

Results

A‐SAA significantly enhanced the time‐ and dose‐dependent expression of ICAM‐1 and VCAM‐1 as effectively as interleukin‐1β/tumor necrosis factor α. A‐SAA promoted the adhesion of PBMCs to FLS and HMVECs. In addition, A‐SAA at 10 μg/ml and 50 μg/ml significantly increased endothelial cell tube formation by 69% and 207%, respectively. At 50 μg/ml and 100 μg/ml, A‐SAA increased HMVEC migration by 188 ± 54% and 296 ± 71%, respectively (mean ± SEM). A‐SAA–induced expression of VCAM‐1, ICAM‐1, and MMP‐1 was down‐regulated by NF‐κB inhibition. Furthermore, A‐SAA induced IκBα degradation and NF‐κB translocation, suggesting that its proinflammatory effects are mediated in part by NF‐κB signaling.

Conclusion

Our findings demonstrate the ability of A‐SAA to induce adhesion molecule expression, angiogenesis, and matrix degradation, mechanisms that are mediated by NF‐κB. Targeting A‐SAA and its signaling pathways may represent a new therapeutic approach in the treatment of RA.

     

(Z)2-(5-(4-methoxybenzylidene)-2, 4-dioxothiazolidin-3-yl) acetic acid protects rats from CCl(4) -induced liver injury

Background and Aim: (Z)2‐(5‐(4‐methoxybenzylidene)‐2, 4‐dioxothiazolidin‐3‐yl) acetic acid (MDA) is an aldose reductase (AR) inhibitor. Recent studies suggest that AR contributes to the pathogenesis of inflammation by affecting the nuclear factor κB (NF‐κB)‐dependent expression of cytokines and chemokines and therefore could be a novel therapeutic target for inflammatory pathology. The current study evaluated the in vivo role of MDA in protecting the liver against injury and fibrogenesis caused by CCl₄ in rats, and the underlying mechanisms. Methods: A single injection of CCl₄ induced acute hepatitis, and repeated injections were used to induce hepatic fibrosis in rats. Therapeutic efficacy was assessed by comparison of the severity of hepatic injury and fibrosis in MDA ‐ treated rats versus untreated controls. Results: MDA significantly protected the liver from injury by reducing the activity of serum alanine aminotransferase, and improving the histological architecture of the liver. MDA modulated NF‐κB‐dependent activation of inflammatory cytokines by reducing hepatic mRNA levels of tumor necrosis factor‐α, interleukin‐1β, inducible nitric oxide (NO) synthase and transforming growth factor‐β. In addition, MDA attenuated oxidative stress by increasing the content of hepatic glutathione. These favorable changes were associated with suppressed hepatic NF‐κB activation by MDA. MDA treatment improved liver fibrosis in rats that received repeated CCl₄ injections. In vitro, MDA attenuated phosphorylation of IκB and activation of NF‐κB, and thus prevented biosynthesis of NO in lipopolysaccharide‐activated RAW264.7 cells. Conclusions: The present study suggests that AR is a novel therapeutic anti‐inflammatory target for the treatment of hepatitis and liver fibrosis.