Nuclear Translocation Of AP-1 Research Articles

Multi-target regulation of pro-inflammatory cytokine production by transcription factor Blimp-1

ObjectiveCytokine storm syndrome is a fatal condition related to infectious and autoimmune diseases. Here, we aim to investigate the regulatory mechanisms of Blimp-1 on multiple cytokine production.MethodsThe Blimp1 shRNA was transfected into RAW264.7 macrophages, followed by Toll-like receptor (TLR) ligand stimulation. The mRNA and protein levels of cytokines were detected by real-time PCR and flow cytometric bead array. The nuclear translocation of AP-1 and NF-κB p65 was measured by immunofluorescence staining. The transcriptional activity was detected by luciferase reporter assay with 5 × NF-κB reporter or with IL6 promoter reporter.ResultsBlimp-1 significantly inhibited the expression and secretion of IL-1β, IL-6, and IL-18 in macrophages during stimulation with a variety of TLR ligands. The immunofluorescence staining results showed that Blimp-1 strictly controlled the nuclear translocation of NF-κB p65 in LPS-challenged macrophages. Furthermore, Blimp-1 directly inhibited the transcriptional activity of NF-κB and the transcription of IL6 gene.ConclusionBlimp-1 represses the production of multiple pro-inflammatory cytokines by directly binding the genomic region and restricting the nuclear translocation and transcriptional activity of NF-κB. This finding may provide potential therapeutic strategies for the cytokine storm-related diseases.

Stanniocalcin-1 mediates negative regulatory action of epidermal layer on expression of matrix-related genes in dermal fibroblasts.

Dermal–epidermal interaction plays a role in many pathophysiological processes, such as tumor invasion and psoriasis, as well as wound healing, and is mediated at least in part by secretory factors. In this study, we investigated the factor(s) involved. We found that stanniocalcin‐1 (STC1), a cytokine, is expressed at the basal layer of epidermis. Knockdown of STC1 with siRNA in HaCaT cells decreased matrix metalloproteinase 1 (MMP1) expression, suggesting that STC1 serves as an autocrine factor, maintaining MMP1 mRNA expression in the epidermal layer. In dermal fibroblasts, STC1 increased MMP1 mRNA expression and decreased collagen1A1 and elastin mRNA expression. These actions were inhibited by SP600125, a jun kinase (JNK) inhibitor. Nuclear translocation of AP‐1, a downstream signal of JNK, was implicated in the actions of STC1. In a coculture system of HaCaT cells and fibroblasts, used as a model of dermal–epidermal interaction, knockdown of STC1 in HaCaT cells with siRNA reduced the negative effects (i.e., induction of MMP1 and decrease of collagen1A1 and elastin) of STC1 on fibroblasts. These results suggest that STC1 secreted from the epidermal layer is a mediator of dermal–epidermal interaction.

Schisandra Chinensis Lignans Suppresses the Production of Inflammatory Mediators Regulated by NF-κB, AP-1, and IRF3 in Lipopolysaccharide-Stimulated RAW264.7 Cells.

Schisandra Fructus (SF) is a traditional Chinese herb used in the treatment of inflammatory disorders like hepatitis. One of the main anti-inflammatory components of SF is the lignans. However, the underlying anti-inflammatory mechanism of Schisandra Chinensis lignans (SCL) remains unclear. This study aims to investigate the effects of SCL on inflammatory mediators in lipopolysaccharide-stimulated RAW264.7 cells and explore the underlying mechanism. The production of nitric oxide (NO) was determined by Griess reaction. ELISA was used to determine cytokine levels and chemokines secretion. To estimate protein levels and enzyme activities, we employed Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was detected using immunofluorescence analyses. The results showed that SCL significantly reduced the release of inflammatory mediators, including NO and PGE2, which may be related to down-regulation of iNOS and COX-2 expression. The production of cytokines and chemokines was suppressed by SCL treatment. SCL also decreased the phosphorylation of IKKα/β, IκB-α, Akt, TBK1, ERK, p38, JNK, NF-κB (p65), AP-1 (c-Jun), and IRF3 in RAW264.7 macrophages activated with LPS. The nuclear protein levels and nuclear translocation of AP-1, NF-κB and IRF3 were suppressed by SCL. These results indicated that SCL suppressed the IKKα/β/NF-κB, MAPKs/AP-1 and TBK1/IRF3 signaling pathways in LPS-stimulated RAW264.7 macrophages.

LL202 protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting MAPK/AP-1 signaling.

LL202, a newly-synthesized flavonoid derivative, has been reported to inhibit inflammatory-induced angiogenesis. However, the exact role of LL202 in inflammation along with its mechanism has not been explored. In this study, we investigated the anti-inflammatory effect of LL202 on intestinal inflammation by establishing dextran sulfate sodium (DSS)-induced experimental colitis. LL202 attenuated DSS-induced body weight loss, colon length shortening and colonic pathological damage. The inflammatory cells infiltration, myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities were decreased by LL202 in a dose-dependent manner. LL202 reduced the production of pro-inflammatory cytokines in serum and colon of DSS-induced mice as well. Mechanically, LL202 could decrease the expression and nuclear translation of AP-1 to protect against DSS-induced colitis. In lipopolysaccharide (LPS)-induced THP-1 cells, LL202 markedly decreased the secretion, mRNA level and protein expression of IL-1β, IL-6 and TNF-α via inhibiting ERK/JNK/p38 MAPK pathways and the nuclear translocation of AP-1. Furthermore, these findings were confirmed in LPS-induced bone marrow derived macrophages (BMDM). In conclusion, our study demonstrated that LL202 could exert its anti-inflammatory effect via inhibiting MAPK/AP-1 signaling, which suggested that LL202 might be a potential effective drug for the treatment of inflammatory bowel diseases.

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes

In inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation.

Black rice anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA expression

Tumor metastasis is the most important cause of cancer death and various treatment strategies have targeted on preventing the occurrence of metastasis. Anthocyanins are natural colorants belonging to the flavonoid family, and are wildly used for their antioxidant properties. Here, we provided molecular evidence associated with the anti-metastatic effects of peonidin 3-glucoside and cyanidin 3-glucoside, major anthocyanins extracted from black rice ( Oryza sativa L. indica), by showing a marked inhibition on the invasion and motility of SKHep-1 cells. This effect was associated with a reduced expression of matrix metalloproteinase (MMP)-9 and urokinase-type plasminogen activator (u-PA). Peonidin 3-glucoside and cyanidin 3-glucoside also exerted an inhibitory effect on the DNA binding activity and the nuclear translocation of AP-1. Furthermore, these compounds also exerted an inhibitory effect of cell invasion on various cancer cells (SCC-4, Huh-7, and HeLa). Finally, anthocyanins from O. sativa L. indica (OAs) were evidenced by its inhibition on the growth of SKHep-1 cells in vivo.

NF‐κB and AP‐1 differentially contribute to the induction of Mn‐SOD and eNOS during the development of oxidant tolerance

Exposure of cardiac myocytes to anoxia/reoxygenation (A/R) increases myocyte oxidant stress and converts the myocytes to a proinflammatory phenotype. These oxidant-induced effects are prevented by pretreatment of the myocytes with an oxidant stress (A/R or H2O2) 24 h earlier (oxidant tolerance). Although NF-kappaB and AP-1 (nuclear signaling) and Mn-SOD and eNOS (effector enzymes) have been implicated in the development oxidant tolerance, the precise relationship between the nuclear transcription factors and the effector enzymes in the development of oxidant tolerance has not been defined. Herein, we show that an initial A/R challenge results in nuclear accumulation of both NF-kappaB and AP-1 (EMSA). In addition, blockade of nuclear translocation of NF-kappaB (SN50) or AP-1 (decoy oligonucleotide) prevents the development of oxidant tolerance, i.e., the second A/R challenge produces the same quantitative effects as the initial A/R challenge. In this model, nuclear translocation of both NF-kappaB and AP-1 is required for induction of Mn-SOD, while nuclear translocation of AP-1, but not NF-kappaB, is a prerequisite for induction of eNOS. Collectively, our findings indicate that NF-kappaB and AP-1 work in concert to ensure the induction eNOS and Mn-SOD, which in turn are important for the development of oxidant tolerance.

Differential involvement of phosphoinositide 3-kinase/Akt in human RPE MCP-1 and IL-8 expression.

To investigate the role of the phosphatidylinositol 3-kinase (PI3K) pathway and the signal mediator AP-1 in monocyte chemotactic protein (MCP)-1 and interleukin (IL)-8 gene expression in human retinal pigment epithelial (hRPE) cells. hRPE cells were stimulated with IL-1beta and TNF-alpha and by coculturing with monocytes in the presence or absence of a series of kinase inhibitors. The induction of MCP-1 and IL-8 protein and mRNA was determined by ELISA and RT-PCR, respectively. Western blot analysis, kinase assays, and electrophoretic mobility shift assays were used to detect the activation of signaling mediators and transcription factors. Concomitant with the induction of chemokine expression by the stimuli, there was phosphorylation of PI3K and its downstream targets-namely, Akt, GSK, and FKHR. Ly294002, a specific inhibitor of PI3K, resulted in time- and dose-dependent blockade of MCP-1 mRNA expression and protein production. The IC(50) for inhibition of MCP-1 secretion induced by IL-1beta, TNF-alpha, and hRPE-monocyte binding was 16, 12, and less than 3 micro M, respectively. In contrast, Ly294002 did not inhibit the IL-8 expression induced by any of the stimuli. Ly294002 as well as U0126, SB202190, and SP600125, the selective inhibitors of MEK, p38, and JNK, respectively, strongly inhibited induced c-fos expression, whereas Ly294002 did not inhibit induction of MEK, p38, or JNK. Blockade of PI3K/Akt abolished IL-1beta-induced nuclear translocation of AP-1, whereas the induction of IkappaB degradation was unchanged. The Ly294002-sensitive PI3K/Akt pathway regulates MCP-1, but not IL-8 expression in hRPE cells independent of MAPK and IkappaB. PI3K-dependent induction of hRPE c-fos and AP-1 nuclear translocation may be a target for therapies aimed at modulating MCP-1 in retinal diseases.

Slow channel calcium inhibition blocks proinflammatory gene signaling and reduces macrophage responsiveness.

This study investigates the possible intracellular mechanisms responsible for calcium antagonist protection in tissue-fixed macrophages, a central modulator of the proinflammatory phenotype. Rabbit alveolar macrophages were exposed to lipopolysaccharide in the presence of different specific calcium antagonists. Cellular and nuclear protein were extracted and analyzed by Western blot for the phosphorylated forms of PYK2, ERK 1/2, and p38, and nuclear translocation of NF-kappaB and AP-1. Tumor necrosis factor-alpha (TNF-alpha) expression was measured by an L929 bioassay on cellular supernatants. Statistical analysis was performed by unpaired Student's t tests. Cells pretreated with 100 to 500 micromol/L of diltiazem or 50 to 100 micromol/L of verapamil, both slow channel calcium blockers, led to dose-dependent reductions in lipopolysaccharide-induced PYK2 and ERK 1/2 phosphorylation, and nuclear translocation of AP-1 when compared with controls (p < 0.05). Neither inhibitor had any significant effect on p38 or NF-kappaB translocation. EGTA an extracellular calcium chelator, had no significant effect on any intracellular process studied. A dose-dependent reduction in TNF-alpha production was demonstrated with diltiazem and verapamil (p < 0.05), with no effect induced by EGTA. Slow channel calcium influx is essential for optimal intracellular signaling through PYK2 and ERK 1/2. This reduced intracellular signaling correlated with reduced AP-1 translocation and TNF-alpha production. Extracellular calcium chelation had no significant effect on intracellular signaling or TNF-alpha production. This study further elucidates the protective mechanism of action of calcium channel blockade by diltiazem and verapamil by reducing intracellular calcium release and down-regulating the excessive proinflammatory phenotype.