Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

Interleukin-1beta induces macrophage inflammatory protein-1beta expression in human hepatocytes

The investigation of factors that regulate expression of CC-chemokines, the important mediators in immune responses and inflammation processes, has an important significance in understanding the immunopathogenesis of liver diseases. We examined the role of interleukin-1beta (IL-1beta), a multifunctional cytokine, in regulating the expression of macrophage inflammatory protein (MIP)-1beta in human hepatocytes (Huh7 and HepG2). IL-1beta significantly enhanced MIP-1beta expression in these cells at both the mRNA and protein levels. Cytokine-enriched supernatants from monocyte-derived macrophage (MDM) cultures also induced MIP-1beta expression. IL-1beta is responsible for MDM supernatant-mediated up-regulation of MIP-1beta since the antibody to IL-1beta abolished MDM supernatant action. Investigation of the mechanism involved in MIP-1beta induction by IL-1beta showed that IL-1beta activated the nuclear factor kappa B (NF-kappaB) promoter in Huh7 cells. In addition, caffeic acid phenethyl ester (CAPE), a specific inhibitor of the activation of NF-kappaB, not only abolished IL-1beta-mediated NF-kappaB promoter activation, but also blocked IL-1beta-induced MIP-1beta expression. These observations suggest that IL-1beta-mediated up-regulation of MIP-1beta production in the hepatic cells may contribute a critical mechanism for continuous recruitment of inflammatory cell to liver and maintenance of inflammation.     

Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination

Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-kappaB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-kappaB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-kappaB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70DeltaC(1-428aa) with N-terminal ATPase domain and Hsp70C(428-642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-kappaB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-kappaB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

NF-kappaB inhibition by an adenovirus expressed aptamer sensitizes TNFalpha-induced apoptosis

Prolonged activation of NF-kappaB is involved in the pathogenesis of chronic inflammatory diseases and associated cancers. NF-kappaB activation is considered to be a main mechanism opposing TNFalpha-induced apoptosis. We investigated whether inhibition of NF-kappaB could sensitize tumor and endothelial cells to TNFalpha-induced apoptosis. As such, we developed a novel H1 RNA polymerase III promoter driven adenoviral vector to express an RNA aptamer, Ad-A-p50, which selectively inhibits NF-kappaB activation in the nucleus. This event sensitizes human lung adenocarcinoma cells (A549) and human endothelial cells (HUVEC) to TNFalpha-induced apoptosis through the multiple pathways regulated by NF-kappaB, including Bcl-XL, HIF-1alpha, and VEGF. Our findings also suggest a new mechanism of HIF-1alpha regulation by NF-kappaB in the normoxic environment. RNA aptamer inhibition of NF-kappaB offers exciting opportunities for sensitizing inflammatory and tumor cells to TNFalpha-induced apoptosis.     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

Hypertonic sodium chloride and mannitol induces COX-2 via different signaling pathways in mouse cortical collecting duct M-1 cells

The kidney cortical collecting duct is an important site for the maintenance of sodium balance. Previous studies have shown that, in renal medullary cells, hypertonic stress induces expression of cyclooxygenase-2 (COX-2) via NF-kappaB activation, but little is known about COX-2 expression in response to hypertonicity in the cortical collecting duct. Therefore, we examined the mechanism of hypertonic induction of COX-2 in M-1 cells derived from mouse cortical collecting duct. Induction of COX-2 protein was detected within 6 h of treatment with hypertonic sodium chloride. The treatment also increased COX-2 mRNA accumulation in a cycloheximide-independent manner, suggesting that ongoing protein synthesis is not required for COX-2 induction. Using reporter plasmids containing 0.2-, 0.3-, and 1.5-kb fragments of the COX-2 promoter, we found that hypertonic induction of COX-2 was due to an increase in promoter activity. The COX-2-inductive effect of hypertonicity was inhibited by SB203580, indicating that the effect is mediated by p38 MAPK. Since p38 MAPK can activate NF-kappaB, we made point mutations in the NF-kappaB binding site within the COX-2 promoter. The mutations did not block the induction of COX-2 promoter activity by hypertonic sodium chloride, and hypertonic sodium chloride failed to activate NF-kappaB binding site-driven reporter gene constructs. In contrast, hypertonic mannitol activated NF-kappaB, indicating that hypertonic mannitol and hypertonic sodium chloride activate COX-2 by different mechanisms. Thus, induction of COX-2 expression in M-1 cells by hypertonic sodium chloride does not involve activation of NF-kappaB. Furthermore, the signal transduction pathways that respond to hypertonic stress vary for different osmolytes in cortical collecting duct cells.     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.