Cell migration to CXCL12 requires simultaneous IKKα and IKKβ-dependent NF-κB signaling

CXCL12 and its unique receptor CXCR4, is critical for the homing of a variety of cell lineages during both development and tissue repair. CXCL12 is particularly important for the recruitment of hemato/lymphopoietic cells to their target organs. In conjunction with the damage-associated alarmin molecule HMGB1, CXCL12 mediates immune effector and stem/progenitor cell migration towards damaged tissues for subsequent repair. Previously, we showed that cell migration to HMGB1 simultaneously requires both IKKβ and IKKα-dependent NF-κB activation. IKKβ-mediated activation maintains sufficient expression of HMGB1's receptor RAGE, while IKKα-dependent NF-κB activation ensures continuous production of CXCL12, which complexes with HMGB1 to engage CXCR4. Here using fibroblasts and primary mature macrophages, we show that IKKβ and IKKα are simultaneously essential for cell migration in response to CXCL12 alone. Non-canonical NF-κB pathway subunits RelB and p52 are also both essential for cell migration towards CXCL12, suggesting that IKKα is required to drive non-canonical NF-κB signaling. Flow cytometric analyses of CXCR4 expression show that IKKβ, but not IKKα, is required to maintain a critical threshold level of this CXCL12 receptor. Time-lapse video microscopy experiments in primary MEFs reveal that IKKα is required both for polarization of cells towards a CXCL12 gradient and to establish a basal level of velocity towards CXCL12. In addition, CXCL12 modestly up-regulates IKKα-dependent p52 nuclear translocation and IKKα-dependent expression of the CXCL12 gene. On the basis of our collective results we posit that IKKα is needed to maintain the basal expression of a critical protein co-factor required for cell migration to CXCL12.     

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-α through IL-12/TNF-α/NF-κB autocrine loop

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor (TNF-). Mo-DCs-Tum showed higher secretions of IL-12 and TNF- than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-B) activation was also induced in Mo-DCs-Tum within 6 h. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF- secretions from Mo-DCs-Tum. Administration of recombinant TNF- or IL-12 enhanced IL-12 or TNF- secretion respectively in Mo-DCs-Tum. Addition of anti-TNF- or anti-IL-12 neutralizing antibody decreased NF-B activation and IL-12 or TNF- secretion in Mo-DCs-Tum. These results suggest that TNF- or IL-12 secretion induces NF-B activation, and it stimulates further TNF- and IL-12 secretions, i.e., an IL-12/TNF-/NF-B autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF- through accelerated NF-B activation induced by the IL-12/TNF-/NF-B autocrine loop.     

Chlorogenic acid inhibits proliferation in human hepatoma cells by suppressing noncanonical NF-κB signaling pathway and triggering mitochondrial apoptosis

Chlorogenic acid (CGA), a phenylpropanoid derived from Eucommia ulmoides Oliver, has been shown to exhibit potent cytotoxic and anti-proliferative activities against several human cancers. However, the effects of CGA on hepatocellular carcinoma (HCC) and the underlying mechanisms have not been intensively studied. In this study, the CGA treatment effects on the viability of human hepatoma cells were investigated by MTT assay. Our data showed that CGA could dose-dependently inhibit the activity of human hepatoma cells Hep-G2 and Huh-7, but did not affect the activity and growth of normal human hepatocyte QSG-7701. The genes and pathways influenced by CGA treatment were explored by RNA sequencing and bioinformatics analysis, which identified 323 differentially expressed genes (DEGs) involved in multiple pharmacological signaling pathways such as MAPK, NF-κB, apoptosis and TGF-β signaling pathways. Further analyses by real-time quantitative PCR, Western blot and flow cytometry revealed that CGA effectually suppressed the noncanonical NF-κB signaling pathway, meanwhile it activated the mitochondrial apoptosis of HCC by upregulation of the BH3-only protein Bcl-2 binding component 3 (BBC3). Our findings demonstrated the potential of CGA in suppressing human hepatoma cells and provided a new insight into the anti-cancer mechanism of CGA.

     

Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells

BackgroundThe intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs.MethodsFor these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs.ResultsTreatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls.SummaryThese in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.     

The activation of HMGB1 as a progression factor on inflammation response in normal human bronchial epithelial cells through RAGE/JNK/NF-κB pathway

Extracellular high-mobility group box-1 (HMGB-1) has been implicated in the inflammation response leading to the precancerous lesions of non-small cell lung cancer (NSCLC). However, the role of HMGB-1 in the inflammation response in normal human bronchial epithelial (NHBE) cells and its underlying mechanisms were still not fully understood. In this study, the inflammation response in NHBE cells was stimulated by 2.5, 5, and 10 μg/ml HMGB-1. However, the receptor for advanced glycation end products (RAGE) blocker RAGE-Ab (5 μg/ml) or 10 μM c-Jun N-terminal kinases (JNK) inhibitor SP600125 could inhibit HMGB1-induced the release of inflammation cytokines including TNF-α, IL-8, IL-10, and MCP-1 in a dose-dependent manner. Furthermore, HMGB1-induced RAGE protein expression, JNK and NF-κB activation were attenuated by the pretreatment with RAGE-Ab or JNK inhibitor SP600125 in Western blot analysis. Our data indicated that HMGB-1 induced inflammation response in NHBE cells through activating RAGE/JNK/NF-κB pathway. HMGB-1 could act as a therapeutic target for inflammation leading NHBE cells to the precancerous lesions of NSCLC.     

The LIM-only protein FHL2 regulates interleukin-6 expression through p38 MAPK mediated NF-κB pathway in muscle cells

Interleukin 6 (IL-6) is pleiotropic cytokine playing an important role in inflammatory response. Other than classical immune tissues, IL-6 is also produced in muscle cells under specific conditions. Four-and-a-half LIM-only protein 2 (FHL2) is preferentially expressed in skeletal and cardiac muscle cells compared to other tissues indicating it has an important role in skeletal muscle and cardiovascular system. In this report, the regulation of IL-6 by FHL2 in muscle cells was investigated. We demonstrated that FHL2 overexpression increased IL-6 mRNA level and its protein secretion in skeletal myoblasts. In contrast, the IL-6 secretion was significantly decreased after FHL2-knockdown by siRNA in response to TNFα stimulation. We further showed that FHL2-mediated induction of IL-6 was regulated by the activation of IL-6 promoter through stimulating NF-κB and p38 MAPK signaling pathway. Our results further illustrated the molecular mechanisms of IL-6 production, which provides new insights in the roles of FHL2 in post-injury inflammation or cytoprotection of muscle cells.     

p50 (NF-κB1) is an effector protein in the cytotoxic response to DNA methylation damage

The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.     

Thymic stromal lymphopoietin is expressed and produced by caspase-1/NF-κB pathway in mast cells

Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease. Although there are many reports regarding function and regulatory mechanism of TSLP in dendritic cells and/or T cells, the regulatory mechanism of TSLP in mast cells has not been fully elucidated. Here, we describe how TSLP is expressed and produced by inflammatory stimulus in mast cells. TSLP mRNA was expressed by phorbol myristate acetate (PMA) plus A23187 stimulation in HMC-1 cells and reached its peak 5h after PMA plus A23187 stimulation. The expression of TSLP mRNA was inhibited by nuclear factor (NF)-κB inhibitor. In addition, NF-κB luciferase activity was inhibited by caspase-1 inhibitor, indicating that caspase-1 is an upstream of NF-κB in mast cells. Furthermore, caspase-1 inhibitor decreased the expression of TSLP mRNA induced by PMA plus A23187. Finally, TSLP production was inhibited by both caspase-1 inhibitor and NF-κB inhibitor. These results provide proof of principle that TSLP can be expressed and produced through caspase-1 and NF-κB in mast cells and open new perspectives to pharmacologically manipulate the expression and production of TSLP by molecules acting on the caspase-1 and NF-κB pathway.     

Plexin-B1 activates NF-κB and IL-8 to promote a pro-angiogenic response in endothelial cells

Background

The semaphorins and their receptors, the plexins, are proteins related to c-Met and the scatter factors that have been implicated in an expanding signal transduction network involving co-receptors, RhoA and Ras activation and deactivation, and phosphorylation events. Our previous work has demonstrated that Semaphorin 4D (Sema4D) acts through its receptor, Plexin-B1, on endothelial cells to promote angiogenesis in a RhoA and Akt-dependent manner. Since NF-κB has been linked to promotion of angiogenesis and can be activated by Akt in some contexts, we wanted to examine NF-κB in Sema4D treated cells to determine if there was biological significance for the pro-angiogenic phenotype observed in endothelium.

Methods/Principal Findings

Using RNA interference techniques, gel shifts and NF-κB reporter assays, we demonstrated NF-κB translocation to the nucleus in Sema4D treated endothelial cells occurring downstream of Plexin-B1. This response was necessary for endothelial cell migration and capillary tube formation and protected endothelial cells against apoptosis as well, but had no effect on cell proliferation. We dissected Plexin-B1 signaling with chimeric receptor constructs and discovered that the ability to activate NF-κB was dependent upon Plexin-B1 acting through Rho and Akt, but did not involve its role as a Ras inhibitor. Indeed, inhibition of Rho by C3 toxin and Akt by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked Sema4D-mediated endothelial cell migration and tubulogenesis. We also observed that Sema4D treatment of endothelial cells induced production of the NF-κB downstream target IL-8, a response necessary for angiogenesis. Finally, we could show through co-immunofluorescence for p65 and CD31 that Sema4D produced by tumor xenografts in nude mice activated NF-κB in vessels of the tumor stroma.

Conclusion/Significance

These findings provide evidence that Sema4D/Plexin-B1-mediated NF-κB activation and IL-8 production is critical in the generation a pro-angiogenic phenotype in endothelial cells and suggests a new therapeutic target for the anti-angiogenic treatment of some cancers.     

DJ-1 activates the noncanonical NF-κB pathway via interaction with Cezanne to inhibit the apoptosis and promote the proliferation of Ishikawa cells

Molecular Biology Reports - Endometrial cancer is generally one of the most evident malignant tumours of the female reproductive system, and the mechanisms underlying its cell proliferation and...