Parkin interacts with the proteasome subunit alpha4

Mutations in the parkin gene encoding an E3 ligase are responsible for autosomal recessive Parkinson's disease. Putative parkin substrates and interacting partners have been identified, but the molecular mechanism underlying parkin-related neurodegeneration is still unclear. We have identified the 20S proteasomal subunit alpha4 (synonyms: PSMA7, XAPC7, subunit alpha type 7) as a new interacting partner of parkin. The C-terminal IBR-RING domain of parkin and the C-terminal part of alpha4 were essential for the interaction. Biochemical studies revealed that alpha4 was not a substrate for parkin-dependent ubiquitylation. Putative functions of the interaction might therefore be substrate presentation to the proteasome or regulation of proteasomal activity. Full-length parkin and parkin lacking the N-terminal ubiquitin-like domain slightly increased the proteasomal activity in HEK 293T cells, in line with the latter hypothesis.     

Ca2+/calmodulin-dependent protein phosphatase calcineurin mediates the expression of iNOS through IKK and NF-kappaB activity in LPS-stimulated mouse peritoneal macrophages and RAW 264.7 cells

Ca(2+) and Ca(2+)/calmodulin-dependent protein phosphatase calcineurin (CN) have been known to play crucial roles in immune response and inflammation. Using mouse peritoneal macrophages and RAW 264.7 macrophage cells, we demonstrated that LPS mobilized intracellular free Ca(2+) and induced CN phosphatase activity. iNOS expression and NO secretion in response to LPS were suppressed by Ca(2+) antagonists (TMB-8, BAPTA/AM, and nifedipine) and CN inhibitor (cyclosporin A). Transient expression of constitutively active CN in mouse peritoneal macrophages and RAW 264.7 macrophages strongly activated NF-kappaB, a key mediator of iNOS expression. We also found that CN mediates NF-kappaB activation via IkappaB-alpha hyperphosphorylation and degradation. Overexpression of dominant negative mutant of IKKalpha and -beta demonstrates that only IKKbeta is the target for CN. These results indicate that CN is required for full iNOS expression and the effective activation of NF-kappaB in RAW 264.7 and peritoneal macrophages.     

Angiopoietin-1/Tie2 signaling pathway inhibits lipopolysaccharide-induced activation of RAW264.7 macrophage cells

Angiopoietin-1 (Ang1) is a ligand for the endothelial-specific tyrosine kinase receptor Tie2 and has been shown to play an essential role in embryonic vasculature development. There have been many studies about the anti-inflammatory effects of Ang1, most of which focus on endothelium cells. In the present study, we explore the role of Ang1-Tie2 signaling in the activation of macrophages upon lipopolysaccharide (LPS) stimulation. We found that Tie2 receptor is expressed on macrophages and Ang1 could inhibit LPS-induced activation of macrophages, as evidenced by cell migration and TNF-α production, specifically through Tie2 receptor. We further investigated the mechanism and found that Ang1-Tie2 could block LPS-induced activation of NF-κB which has been shown to be necessary for macrophage activation with LPS treatment. Thus, we described, for the first time, the role of Ang1-Tie2 signaling in macrophage activation and the possible mechanisms in response to immune stimulation.     

The immunostimulatory effect of indole-6-carboxaldehyde isolated from Sargassum thunbergii (Mertens) Kuntze in RAW 264.7 macrophages

ABSTRACT

Indole-6-carboxaldehyde (I6CA), an indole derivative isolated from the marine brown algae Sargassum thunbergii, is known to have several beneficial effects, but no studies on immune regulation have been conducted. In this study, the immunomodulatory properties of I6CA on murine RAW 264.7 monocyte/macrophage cells were evaluated. As the concentration of I6CA increased, the morphology of RAW 264.7 cells changed to a typical active macrophage shape, and the phagocytic activity increased significantly. I6CA effectively enhanced the production and secretion of immunomodulatory mediators and cytokines due to increased expression of their respective genes. Additionally, I6CA markedly stimulated the expression of Toll-like receptor 4 (TLR4) and its adapter molecule, myeloid differentiation factor 88 (Myd88), and increased TLR4 complexed with Myd88. Furthermore, I6CA promoted the nuclear translocation of nuclear factor-kappa B (NF-κB) by increasing the degradation of the inhibitor of NF-κB-α. Meanwhile, similar trends were also found in lipopolysaccharide-treated cells as a positive control. Furthermore, molecular docking simulation showed that I6CA interacted with TLR4-myeloid differentiation 2 complex. Taken together, the results support the concept that I6CA may increase the activity of the TLR4/NF-κB signaling pathway in order to enhance the immunomodulatory activity of RAW 264.7 cells.     

Double stranded RNA-dependent protein kinase is involved in osteoclast differentiation of RAW264.7 cells in vitro

Double-stranded RNA-dependent protein kinase (PKR) plays a critical role in antiviral defence of the host cells. PKR is also involved in cell cycle progression, cell proliferation, cell differentiation, tumorigenesis, and apoptosis. We previously reported that PKR is required for differentiation and calcification of osteoblasts. However, it is unknown about the role of PKR in osteoclast differentiation. A dominant-negative PKR mutant cDNA, in which the amino acid lysine at 296 was replaced with arginine, was transfected into RAW264.7 cells. We have established the cell line that stably expresses the PKR mutant gene (PKR-K/R). Phosphorylation of PKR and α-subunit of eukaryotic initiation factor 2 was not stimulated by polyinosic-polycytidylic acid in the PKR-K/R cells. RANKL stimulated the formation of TRAP-positive multinuclear cells in RAW264.7 cells. However, TRAP-positive multinuclear cells were not formed in the PKR-K/R cells even when the cells were stimulated with higher doses of RANKL. A specific inhibitor of PKR, 2-aminopurine, also suppressed the RANKL-induced osteoclast differentiation in RAW264.7 cells. The expression of macrophage fusion receptor and dendritic cell-specific transmembrane protein significantly decreased in the PKR-K/R cells by real time PCR analysis. The results of RT-PCR revealed that the mRNA expression of osteoclast markers (cathepsin K and calcitonin receptor) was suppressed in the PKR-K/R cells and RAW264.7 cells treated with 2-aminopurine. Expression of NF-κB protein was suppressed in the PKR-K/R cells and 2-aminopurine-treated RAW264.7 cells. The level of STAT1 protein expression was elevated in the PKR-K/R cells compared with that of the wild-type cells. Immunohistochemical study showed that PKR was localized in osteoclasts of metatarsal bone of newborn mouse. The finding that the PKR-positive multinuclear cells should be osteoclasts was confirmed by TRAP-staining. Our present study indicates that PKR plays important roles in the differentiation of osteoclasts.     

Synergistic interaction of proteasome and topoisomerase II inhibition in multiple myeloma

Multiple myeloma is a malignancy of terminally differentiated plasma cells and is incurable in the majority of the patients. Thus, novel effective treatment regimens are urgently needed. In this study, we examined the effects of co-treatment with proteasome-inhibitor bortezomib and topoisomerase II inhibitor etoposide in multiple myeloma cells lines OPM-2, RPMI-S and NCI-H929. Using the median effect method of Chou and Talalay, we evaluated the combination indices (CI) for simultaneous and sequential treatment schedules. In the sequential treatment schedule, we found strong synergistic effects in all three cell lines, even at low single-agent cytotoxicity levels. When cells were treated simultaneously with both drugs, the synergy was present but less pronounced than in the sequential treatment schedule. The synergistic effects observed in the co-treatment schedules were accompanied by an inhibition of anti-apoptotic effects that were induced by etoposide alone. Namely, bortezomib abrogated both etoposide-induced NF-κB activation and etoposide-induced bcl-2 up-regulation. Our data suggest that combining etoposide with bortezomib might be useful for cancer treatment, as bortezomib potentially inhibits counter-regulatory mechanisms of tumor cells, which are induced by topoisomerase II inhibition and which may contribute to acquired chemoresistance.     

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.     

MK615 attenuates Porphyromonas gingivalis lipopolysaccharide-induced pro-inflammatory cytokine release via MAPK inactivation in murine macrophage-like RAW264.7 cells

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-κB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.     

Acetylbritannilatone suppresses NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression

In order to elucidate the mechanism of anti-inflammatory effect of 1-o-acetylbritannilatone (ABL) isolated from Inula Britannica-F, we investigated ABL for its ability to inhibit the inflammatory factor production in RAW 264.7 macrophages. The studies showed that ABL not only inhibited LPS/IFN-gamma-mediated nitric oxide (NO) production and inducible nitric synthase (iNOS) expression, but also decreased LPS/IFN-gamma-induced prostaglandin E2 (PGE2) production and cyclo-oxygenase-2 (COX-2) expression in a concentration-dependent manner. EMSA demonstrated that ABL inhibited effectively the association of NF-kappaB, which is necessary for the expression of iNOS and COX-2, with its binding motif in the promoter of target genes. These data suggest that ABL suppress NO and PGE2 synthesis in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 gene expression, respectively. The anti-inflammatory effect of ABL involves blocking the binding of NF-kappaB to the promoter in the target genes and inhibiting the expression of iNOS and COX-2.     

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.     

ST2 suppresses IL-6 production via the inhibition of IkappaB degradation induced by the LPS signal in THP-1 cells

LPS induces the production of inflammatory cytokines via the stimulation of Toll-like receptors. In this study, we demonstrated that a soluble secreted form of the ST2 gene product (ST2), a member of the interleukin-1 receptor family, suppressed the production of IL-6 in an LPS-stimulated human monocytic leukemia cell line, THP-1. Immunofluorescence confocal microscopy revealed the binding of ST2 to the surface of the THP-1 cells, in which ST2 led to decreased binding of nuclear factor-kappaB to the IL-6 promoter. Furthermore, the degradation of IkappaB in the cytoplasm after LPS stimulation was reduced by pretreatment with ST2. These results demonstrated that ST2 negatively regulates LPS-induced IL-6 production via the inhibition of IkappaB degradation in THP-1 cells.     

Inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by butein in RAW 264.7 cells

Butein has been reported to exert anti-inflammatory effect but the possible mechanism involved is still unclear. Here, we report the inhibitory effect of butein on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) gene expression. Butein also inhibited the induction of tumor necrosis factor-alpha and cyclooxygenase 2 by LPS. To further investigate the mechanism responsible for the inhibition of iNOS gene expression by butein, we examined the effect of butein on LPS-induced nuclear factor-kappaB (NF-kappaB) activation. The LPS-induced DNA binding activity of NF-kappaB was significantly inhibited by butein, and this effect was mediated through inhibition of the degradation of inhibitory factor-kappaB and phosphorylation of Erk1/2 MAP kinase. Furthermore, increased binding of the osteopontin alphavbeta3 integrin receptor by butein may explain its inhibitory effect on LPS-mediated NO production. Taken together, these results suggest that butein inhibits iNOS gene expression, providing possible mechanisms for its anti-inflammatory action.     

Glycyrrhizin inhibits lipopolysaccharide-induced inflammatory response by reducing TLR4 recruitment into lipid rafts in RAW264.7 cells

Background

The aim of this study was to investigate the effect of glycyrrhizin on LPS-induced endotoxemia in mice and clarify the possible mechanism.

Methods

An LPS-induced endotoxemia mouse model was used to confirm the anti-inflammatory activity of glycyrrhizin in vivo. In vitro, RAW264.7 cells were stimulated with LPS in the presence or absence of glycyrrhizin. The expression of cytokines was determined by ELISA. Toll-like receptor 4 (TLR4) was determined by Western blot analysis. Nuclear factor-kB (NF-κB) and Interferon regulatory factor 3 (IRF3) activation were detected by Western blotting and luciferase assay. Lipid raft staining was detected by immunocytochemistry.

Results

In vivo, the results showed that glycyrrhizin can improve survival during lethal endotoxemia. In vitro, glycyrrhizin dose-dependently inhibited the expression of TNF-α, IL-6, IL-1β and RANTES in LPS-stimulated RAW264.7 cells. Western blot analysis showed that glycyrrhizin suppressed LPS-induced NF-κB and IRF3 activation. However, glycyrrhizin did not inhibit NF-κB and IRF3 activation induced by MyD88-dependent (MyD88, IKKβ) or TRIF-dependent (TRIF, TBK1) downstream signaling components. Moreover, glycyrrhizin did not affect the expression of TLR4 and CD14 induced by LPS. Significantly, we found that glycyrrhizin decreased the levels of cholesterol of lipid rafts and inhibited translocation of TLR4 to lipid rafts. Moreover, glycyrrhizin activated ABCA1, which could induce cholesterol efflux from lipid rafts.

Conclusion

Glycyrrhizin exerts an anti-inflammatory property by disrupting lipid rafts and inhibiting translocation of TLR4 to lipid rafts, thereby attenuating LPS-mediated inflammatory response.

General significance

Learning the anti-inflammatory mechanism of glycyrrhizin is crucial for the anti-inflammatory drug development.     

Regulation of lipopolysaccharide-induced inflammatory response by heat shock protein 27 in THP-1 cells

HSP27 is a member of the small HSP family which has been linked to different signaling pathways regulating critical cellular functions. But the role of HSP27 in LPS-induced inflammatory signaling pathways is still unclear. In the present study, both overexpression and RNA interference experiments indicated that HSP27 increased LPS-induced expression of iNOS and COX-2 and release of NO/PGE2 through enhancing NF-κB but not MAPK activation. The effects of HSP27 on LPS-induced iNOS/COX-2 expression and relative signaling cascade were closely related with the phosphorylation of HSP27. Further studies have shown that HSP27-regulated LPS-induced activation of NF-κB by interacting with TRAF6 and increasing the association of TRAF6-IKKγ. This could be a probable mechanism by which HSP27 modulates LPS-induce inflammatory signaling pathways. Thus, HSP27 may play a potential role in regulating inflammatory responses in immunologic system.     

NUMBL interacts with TRAF6 and promotes the degradation of TRAF6

Tumor necrosis factor-associated factor 6 (TRAF6) is an essential adaptor protein for IL-1R or TLR-mediated NF-κB signaling pathway activation. In previous work we have found NUMBL interacts with TAB2 and negatively regulates NF-κB signaling pathway. Here, we report that NUMBL directly binds to TRAF6 in vivo and in vitro. NUMBL down-regulates TRAF6 protein level and shortens its half-life. Furthermore, knockdown of NUMBL significantly increases endogenous TRAF6 protein level in the cultured cortical neurons. In vivo ubiquitination assays indicate that NUMBL promotes the assembly of K48-linked polyubiquitination chains on TRAF6, but has no significant effect on its K63-linked polyubiquitination. Our results collectively reveal that NUMBL interacts with TRAF6 and promotes the degradation of TRAF6 in vivo, leading to the inhibition of NF-κB signaling pathway.     

Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity

Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.     

Antimicrobial peptide HI-3 from Hermetia illucens alleviates inflammation in lipopolysaccharide-stimulated RAW264.7 cells via suppression of the nuclear factor kappa-B signaling pathway

Hermetia illucens-3 (HI-3), an active insect antimicrobial peptide extracted from H. illucens larvae, exerts antibacterial and anticancer activity. However, the inflammatory effects and their relative molecular mechanisms remain unclear. To explore the inflammatory effects of HI-3, an inflammatory model was induced using 1 ng/mL LPS in RAW264.7 cells. The cell viability and phagocytosis of LPS-stimulated RAW264.7 cells were then detected after HI-3 treatment. Furthermore, the antioxidant activity, the levels of proinflammatory cytokines, and the expression levels of both p65 and inhibitor of nuclear factor kappa B (IκB) were measured. Results showed that HI-3 could inhibit the differentiation, proliferation, phagocytosis, and antioxidant ability, as well as the secretion and messenger RNA expression levels of IL-6, TNF-α, and IL-1β of LPS-induced RAW264.7 cells in a dose-dependent manner. At the same time, the level of the anti-inflammatory cytokine IL-10 was increased after HI-3 treatment. Western blotting results showed that HI-3 suppressed LPS-induced p65 and IκB activation in a dose-dependent manner. Therefore, HI-3 exerts its anti-inflammatory effect by inhibiting the expression of proinflammatory cytokines and the activation of p65 and IκB, which indicated that HI-3 could be a promising therapeutic medicine for inflammation.