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1
Fang, Deyu, and Heeyoung Yang. "Identification of a novel deubiquitinating enzyme DUBS6 that regulates Sirt6-mediated suppression of NF-κB in T cells (88.17)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 88.17. http://dx.doi.org/10.4049/jimmunol.184.supp.88.17.
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Abstract The type III histone deacetylase Sirt6 is known as a regulator of aging in diverse species by regulating NF-kB-dependent gene expression. Here, using an LC-MS/MS approach combined with immunoprecipitation, we have identified a complex of Sirt6-interacting molecules (interactome) that contains about 40 proteins from mouse primary T cells. One of the interactome members is a novel deubiquitinating enzyme (DUB); we thus define this gene as a DUB of Sirt6 (DUBS6). Co-immunoprecipitation and western blotting analysis further validated that DUBS6 interacts with Sirt6 in transiently transfected HEK293 cells as well as in mouse primary T cells. Expression of DUBS6 inhibits Sirt6 ubiquitination and thus stabilizes Sirt6. Unexpectedly, DUBS6 expression, despite increased Sirt6 stability, reversed Sirt6-mediated suppressive function of NF-kB transcription activity. These results suggest that ubiquitination of Sirt6 appears to be required for its functions and that DUBS6 is a novel regulator in Sirt6-mediated NF-kB suppression in T cells.
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Oliveira, Vasco A., Linda Mathews, Danielle Yarde, Xingyu Wang, David Boulware, Lori A. Hazlehurst, Dung-Tsa Chen, Amer Beg, and William S. Dalton. "NF-kB as a Regulator of FA/BRCA Gene Expression in Multiple Myeloma." Blood 110, no. 11 (November 16, 2007): 3508. http://dx.doi.org/10.1182/blood.v110.11.3508.3508.
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Abstract Results to date argue compellingly that disruption of FA/BRCA gene expression plays a pivotal role in human somatic carcinogenesis. Melphalan, a DNA cross-linker, is one of the most widely used and effective drugs in the treatment of multiple myeloma (MM). Although most patients respond to standard and high dose melphalan, eventually patients acquire resistance and develop progressive disease. In 1991, our laboratory reported that acquired resistance in a human myeloma cell line was associated with reduced DNA crosslinks, elevated glutathione levels, and increased radiation survival (Cancer Res. 5:993; 1991). Most recently, we reported that the melphalan-resistant myeloma cell lines, 8226/LR5 and U266/LR6, showed a significant increase in several FA/BRCA genes compared to drug-sensitive cells, and that enhanced interstrand crosslink (ICL) repair via this signaling pathway contributes to acquired drug resistance in melphalan resistant cell lines (Blood 10:698; 2005). Here, we report that IKKa is constitutively phosphorylated in unstimulated 8226/LR5 cells, but not in melphalan-sensitive control cells. The specific phosphorylation of IKKa leads to an increase in basal NF-kB DNA binding activity, and 8226/LR5 cells are found to be markedly sensitive to BMS-345541 (a highly selective inhibitor of IkB) relative to control cells. Importantly, a cytotoxic dose of BMS-345541 induces a dramatic decrease in FA/BRCA gene expression, and a concomitant inhibition of NF-kB DNA binding activity in both 8226/S and 8226/LR5 cells. Furthermore, we show that 8226/LR5 cells experience the highest degree of direct binding between FANCD2 promoter and NF-kB/Rel family members, which, in turn, leads to an increase in basal FANCD2-specific NF-kB activity. Small-interfering RNA (siRNA)-mediated depletion of RelB and p50, but not other NF-kB subunits, in 8226 cells results in impaired NF-kB binding activity, and visible decrease in FANCD2 protein expression. Studies designed to dissect the role of NF-kB in acquired melphalan resistance are in progress, and the results will be presented. Our findings suggest that NF-kB functions as a regulator of FA/BRCA expression, and that this pathway represents a new target for preventing acquired drug resistance in myeloma patients.
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Roh, Eunmiri, Heun-Sik Lee, Jeong-Ah Kwak, Sang Hun Jung, Sang-Bae Han, and Youngsoo Kim. "Novel chalcone JSH 4-4 inhibit NF-kB-regulated inflammatory gene expression via targeting LPS-binding site of MD-2 (98.4)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 98.4. http://dx.doi.org/10.4049/jimmunol.184.supp.98.4.
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Abstract Previous studies have shown that chalcone has beneficial effects with antioxidant and anti-inflammatory activities. However, molecular basis for these effects not yet fully understood. In this study, a chalcone derivative, 2’, 4’-dihydroxy-6’-isopentyloxy chalcone (JSH 4-4) was discovered as an inhibitor of NF-kB activation. To demonstrate molecular target affected by JSH 4-4, we examined a homotypic interaction of TLR4, using Ba/F3 cells stably expressing TLR4-Flag, TLR4-GFP CD14 and MD-2, seperately. As a result, JSH 4-4 inhibited the LPS-induced TLR4 oligomerization. Moreover, we demonstrated that JSH 4-4 inhibit LPS-induced NF-kB activation via targeting LPS-binding site of MD-2. This mechanism of action could contribute to suppressive effect of JSH 4-4 on NF-kB-regulated production of inflammatory mediators such as nitric oxide, COX-2 and cytokines. TLR4-mediated NF-kB signalling occurs following sequential activation of IRAK family and TAK1. JSH 4-4 inhibited LPS-induced phosphorylation of IRAK-1 and TAK1. In addition, JSH 4-4 significantly inhibited IKK activity and phosphorylation of IkBa, resulting in sequential prevention of downstream events, including degradation of IkBs, nuclear translocation of NF-kB p65. In conclusion, JSH 4-4 inhibit NF-kB-regulated inflammatory mediators via targeting the receptor complex MD2-TLR4 in macrophages, thus JSH 4-4 might be considered as a potential agent for the treatment of NF-kB-associated inflammatory diseases.
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Kozloski, Goldi A., Xiaoyu Jiang, Shruti Bhatt, Rita Shaknovich, Ari M. Melnick, and Izidore S. Lossos. "Mirna-181a expression Lead to Longer Animal Survival and Slower Tumor-Growth Rate in Diffuse Large B-Cell Lymphoma Xenograft Models." Blood 124, no. 21 (December 6, 2014): 2963. http://dx.doi.org/10.1182/blood.v124.21.2963.2963.
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Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is subdivided into the germinal center B-like (GCB) and activated B cell-like (ABC) subtypes by gene expression profiling, and these subtypes exhibit different clinical outcomes and signaling pathway deregulations. Compared to the GCB, the ABC-DLBCL subtype displays a more aggressive clinical course and shorter patient survival. Constitutive nuclear factor kappa-B (NF-kB) activity is often associated with the ABC-DLBCL subtype, however recent studies suggest that NF-kB signaling activation is also observed to a lower extent in the GCB-DLBCL subtype (Lina Odqvist et al. 2014). miRNAs have diagnostic and prognostic value in disease classification, and growing evidence implicates miRNAs in tumorigenesis, tumor maintenance, and dissemination through their ability to modulate the expression of critical genes and signaling networks. We previously demonstrated that miRNA-181a expression correlates with longer survival in patients treated with R-CHOP, independent of established clinical and molecular predictors. However, the molecular and cellular mechanisms underlying the association between miRNA-181a expression and improved prognosis in DLBCL patients are currently unknown. Herein we analyzed the role of miRNA-181a in DLBCL pathogenesis. Results:Quantitative RT-PCR analyses demonstrate higher endogenous miRNA-181a levels in centroblasts than in plasmablasts. Concordantly, endogenous miRNA-181a levels were significantly higher in GCB DLBCL cell lines and primary tumors compared with ABC DLBCL. These expression differences could not be attributed to distinct DNA methylation signatures in the miRNA-181a promoters (Chromosomes 1, 9) or regulatory elements as analyzed by Mass Array Sequenom Epityping. In search for putative miRNA-181a targets we identified 5 genes (CARD11, NFKB1A (IKBα), NFKB1 (p105/p50), RELA (p65), and REL (CREL)) within the NF-kB signaling pathway. Analyses of these targets show a decrease in the levels of these proteins and mRNAs in ABC and GCB DLBCL cell lines ectopically expressing miRNA-181a compared with scramble control plasmid. Luciferase reporter analyses encoding the respective wild type or mutated 3′UTR sequences demonstrate direct and specific targeting of these transcripts with the exception of RELA. Analysis of the net effect of miRNA-181a on NF-kB signaling using NF-kB luciferase reporter demonstrate significant decrease in NF-kB signaling. Concordantly, anti-miRNA-181a transfection led to increased NF-kB luciferase reporter activity. Moreover, western blot analyses of cytoplasmic and nuclear fractions showed a decrease in the levels of the transcription factors CREL and p50 in both cellular compartments, a decrease in the binding to DNA at NF-kB binding motifs, and a consequent decrease in NF-kB target gene transcription in the miRNA-181a expressing cells compared with scramble control. Together these studies point to miRNA-181a-mediated repression of NF-kB signaling in DLBCLs. Ectopic miRNA-181a expression led to a decrease in cell proliferation and an increase in cell death in both DLBCL subtypes, but this effect was more pronounced in the ABC DLBCL cell lines. The miRNA-181a-mediated increase in cell apoptosis could not be rescued by BCL2 co-transfection, an anti-apoptotic protein that was previously established as a direct miRNA-181a target. Analyses of miRNA-181a effects in NOD/SCID mice demonstrated that in vivo miRNA-181a induction in GCB and ABC human DLBCL xenografts led to decreased tumor growth and significantly longer animal survival. Notably, survival was prolonged in both GCB and ABC DLBCL bearing animals. Figure 1 Figure 1. Conclusions: miRNA-181a directly suppress the NF-kB signaling pathway and lead to increased tumor cell death in both DLBCL subtypes suggesting that NF-kB deregulation is present in both tumor subtypes. However, the lower miRNA-181a expression level in the ABC DLBCL subtype may contribute to the higher NF-kB signaling activity that is observed in this subtype. Furthermore, our study provides a plausible explanation for the association between high miRNA-181a expression and longer survival of DLBCL patients. Disclosures No relevant conflicts of interest to declare.
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Zhu, Ningxi, Lubing Gu, Harry W. Findley, Kuang-Yueh Chiang, and Muxiang Zhou. "Vitamin K3 Selectively Induces Apoptosis in Acute Lymphoblastic Leukemia Cells with Constitutive Activation of IKKα/NF-kB Activation." Blood 106, no. 11 (November 16, 2005): 859. http://dx.doi.org/10.1182/blood.v106.11.859.859.
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Abstract Although the cytotoxic effect of vitamin K3 (VK3) on human cancer cells has been repeatedly reported, no clear conclusions from either in vitro or in vivo tests have so far been made for VK3 as an anticancer agent due to marked inter-tumor variability of efficacy in response to VK3 treatment. Here, we report that sensitivity of neoplastic cells to VK3-induced killing depends on IKKα expression/NF-kB activation in the cells. We tested the sensitivity to VK3 of 14 leukemic cell lines established from children with acute lymphoblastic leukemia (ALL). The 14 lines were classified into three groups: IKKα +/NF-kB+, IKKα +/NF-kB−, IKKα−/NF-kB−. IKKα +/NFkB+ cell lines that are generally resistant to doxorubicin are more sensitive to VK3 induced cell death than are the IKKα +/NFkB− lines that are usually sensitive to doxorubicin. The median of IC 50 values of VK3 and doxorubicin as tested by WST analysis for IKKα +/NFkB+ cells were 3.92 mM and 1.58 mM, respectively, compared to IKKα +/NFkB− cells (7.3 mM of VK3 and 0.71 mM of doxorubicin, p<0.01, t-test). Assays by testing activation of caspase and cleavage of death substrate PARP as well as flow cytometry showed that apoptosis was induced in a line with high levels of IKKα/NF-kB activation at 2 h after VK3 treatment. In contrast, apoptosis was not induced by VK3 even at 48 h post-treatment in two lines that lack IKKa expression and NF-kB activation. To test if IKKα/NF-kB is a molecular target of VK3 inducing apoptosis in ALL, we examined the expression and activation of IKKα/NF-kB in VK3-treated cells. VK3 specifically reduced IKKα expression and inhibited NF-kB activation, resulting in downregulation of NF-kB-mediated gene expression and apoptosis. These results suggest that inhibition of IKKα/NF-kB signaling pathway is essential for VK3 to induce cell death, and that VK3, a dietary factor with no cytotoxic effect on normal cells, would be a useful adjuvant in the treatment of ALL and other cancer patients whose neoplastic cells express constitutive NF-kB and are resistant to chemotherapy.
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Nakagawa, Masahiro, Munetake Shimabe, Nahoko Nishimoto, Naoko Watanabe-Okochi, Motoshi Ichikawa, Yasuhito Nannya, Yoichi Imai, and Mineo Kurokawa. "AML1/Runx1 Is a Cytoplasmic Attenuator of NF-Kb Signaling: Implication in Pathogenesis and Targeted Therapy of AML1-Related Leukemia." Blood 114, no. 22 (November 20, 2009): 1962. http://dx.doi.org/10.1182/blood.v114.22.1962.1962.
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Abstract Abstract 1962 Poster Board I-985 Introduction: AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results: To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions: We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures: No relevant conflicts of interest to declare.
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Endale, Mehari, Tae-Hwan Kim, Yi-Seong Kwak, Na-Mi Kim, Seung-Hyung Kim, Jae Youl Cho, Bong-Sik Yun, and Man-Hee Rhee. "Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation." Mediators of Inflammation 2017 (2017): 1–13. http://dx.doi.org/10.1155/2017/7250968.
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Torilin, a sesquiterpene isolated from the fruits ofTorilis japonica,has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin’s effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2,P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun) activation and IKK-mediated I-κBαdegradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound’s potential as a candidate anti-inflammatory agent.
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Buhrmann, Constanze, Aranka Brockmueller, Anna-Lena Mueller, Parviz Shayan, and Mehdi Shakibaei. "Curcumin Attenuates Environment-Derived Osteoarthritis by Sox9/NF-kB Signaling Axis." International Journal of Molecular Sciences 22, no. 14 (July 16, 2021): 7645. http://dx.doi.org/10.3390/ijms22147645.
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Inflammation has a fundamental impact on the pathophysiology of osteoarthritis (OA), a common form of degenerative arthritis. It has previously been established that curcumin, a component of turmeric (Curcuma longa), has anti-inflammatory properties. This research evaluates the potentials of curcumin on the pathophysiology of OA in vitro. To explore the anti-inflammatory efficacy of curcumin in an inflamed joint, an osteoarthritic environment (OA-EN) model consisting of fibroblasts, T-lymphocytes, 3D-chondrocytes is constructed and co-incubated with TNF-α, antisense oligonucleotides targeting NF-kB (ASO-NF-kB), or an IkB-kinase (IKK) inhibitor (BMS-345541). Our results show that OA-EN, similar to TNF-α, suppresses chondrocyte viability, which is accompanied by a significant decrease in cartilage-specific proteins (collagen II, CSPG, Sox9) and an increase in NF-kB-driven gene proteins participating in inflammation, apoptosis, and breakdown (NF-kB, MMP-9, Cox-2, Caspase-3). Conversely, similar to knockdown of NF-kB at the mRNA level or at the IKK level, curcumin suppresses NF-kB activation, NF-kB-promotes gene proteins derived from the OA-EN, and stimulates collagen II, CSPG, and Sox9 expression. Furthermore, co-immunoprecipitation assay shows that curcumin reduces OA-EN-mediated inflammation and chondrocyte apoptosis, with concomitant chondroprotective effects, due to modulation of Sox-9/NF-kB signaling axis. Finally, curcumin selectively hinders the interaction of p-NF-kB-p65 directly with DNA—this association is disrupted through DTT. These results suggest that curcumin suppresses inflammation in OA-EN via modulating NF-kB-Sox9 coupling and is essential for maintaining homeostasis in OA by balancing chondrocyte survival and inflammatory responses. This may contribute to the alternative treatment of OA with respect to the efficacy of curcumin.
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Lyu, Qingkang, Magdalena Wawrzyniuk, Victor P. M. G. Rutten, Willem van Eden, Alice J. A. M. Sijts, and Femke Broere. "Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line." International Journal of Molecular Sciences 21, no. 18 (September 4, 2020): 6464. http://dx.doi.org/10.3390/ijms21186464.
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The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.
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Dasgupta, Subhajit, Jackie Eudaly, Ivan Molano, DeAnna Baker, Meagan Mollenhauer, and Gary Gilkeson. "Involvement of estrogen receptor alpha on MCP1 production in C57BL/6 kidney mesangial cells (87.29)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 87.29. http://dx.doi.org/10.4049/jimmunol.184.supp.87.29.
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Abstract Abstract We demonstrate an estrogen independent involvement of ERα in TLR2 mediated expression of MCP1 in mesangial cells. In vitro treatment with the TLR2 ligands Pam3CysSK4, LTA and PGN-SA induced production of MCP1 in mesangial cells isolated from kidneys of female C57BL/6 mice in a dose dependent manner. Treatment with LTA to C57BL/6 mesangial cells was found to activate NF kB and induced its nuclear localization. Interestingly enough, we found LTA treatment induced phosphorylation of ERα in primary mesangial cells and also in SV40 virus transformed B6 mesangial cell line. Nuclear translocation of pERα (Ser118) was found in the LTA treated B6 mesangial cells. ERα selective antagonist MPP and NF kB antagonist TPCA1 attenuated MCP1 production in TLR2 ligand treated mesangial cells suggesting influence of pERα in NF kB dependent MCP1 gene expression. The LTA was found to induce DNA binding activity of NF kB in B6 mesangial cell line in a time dependent manner. Transient transfection with ERα siRNA was also found to reduce expression of MCP1 in Pam3CysSK4 treated mesangial cells. Taken together, our study indicates an unique role of ERα in modulation of TLR2-mediated signals in mesangial cells. Acknowledgement: We are thankful to the Department of Medicine, Division of Rheumatology/Immunology and Ralph V Johnson VA research service, MUSC. The research support was provided by VA research service. We do not have any conflict of interests.
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Yang, Bingyuan, Lisette A. Maddison, Karolina E. Zaborska, Chunhua Dai, Linlin Yin, Zihan Tang, Liqing Zang, David A. Jacobson, Alvin C. Powers, and Wenbiao Chen. "RIPK3-mediated inflammation is a conserved β cell response to ER stress." Science Advances 6, no. 51 (December 2020): eabd7272. http://dx.doi.org/10.1126/sciadv.abd7272.
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Islet inflammation is an important etiopathology of type 2 diabetes; however, the underlying mechanisms are not well defined. Using complementary experimental models, we discovered RIPK3-dependent IL1B induction in β cells as an instigator of islet inflammation. In cultured β cells, ER stress activated RIPK3, leading to NF-kB–mediated proinflammatory gene expression. In a zebrafish muscle insulin resistance model, overnutrition caused islet inflammation, β cell dysfunction, and loss in an ER stress–, ripk3-, and il1b-dependent manner. In mouse islets, high-fat diet triggered the IL1B expression in β cells before macrophage recruitment in vivo, and RIPK3 inhibition suppressed palmitate-induced β cell dysfunction and Il1b expression in vitro. Furthermore, in human islets grafted in hyperglycemic mice, a marked increase in ER stress, RIPK3, and NF-kB activation in β cells were accompanied with murine macrophage infiltration. Thus, RIPK3-mediated induction of proinflammatory mediators is a conserved, previously unrecognized β cell response to metabolic stress and a mediator of the ensuing islet inflammation.
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Friedrichsen, Sönke, Claire V. Harper, Sabrina Semprini, Michael Wilding, Antony D. Adamson, Dave G. Spiller, Glyn Nelson, John J. Mullins, Michael R. H. White, and Julian R. E. Davis. "Tumor Necrosis Factor-α Activates the Human Prolactin Gene Promoter via Nuclear Factor-κB Signaling." Endocrinology 147, no. 2 (February 1, 2006): 773–81. http://dx.doi.org/10.1210/en.2005-0967.
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Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-α activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-α in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-α, Western blot analysis showed rapid inhibitory protein κB (IκBα) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IκBα fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IκBα degradation. This was associated with increased nuclear factor κB (NF-κB)-mediated transcription from an NF-κB-responsive luciferase reporter construct. Disruption of NF-κB signaling by expression of dominant-negative variants of IκB kinases or truncated IκBα abolished TNF-α activation of the prolactin promoter, suggesting that this effect was mediated by NF-κB. TNF-α signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function.
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Le Clorennec, Christophe, Ibtissam Youlyouz-Marfak, Eric Adriaenssens, Jean Coll, Dominique Bordessoule, Georg W. Bornkamm, and Jean Feuillard. "Regulation of CD95 in EBV infected B-Cells." Blood 104, no. 11 (November 16, 2004): 81. http://dx.doi.org/10.1182/blood.v104.11.81.81.
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Abstract Expression of CD95 is regulated by various agents such as TNFa, IFNg, or Daunorubicine, able to induce the transcriptional factors NF-kB, STAT1 and p53 respectively. The CD95 promoter is known to harbour binding sites for these factors. These factors are modulated both in expression and activity during EBV infection of B-cells. EBV is known to induce CD95 expression. The objective of this work was to understand the contribution of p53, STAT1 and NF-kB in the regulation of CD95 expression by EBV. To control the activity of NF-kB, STAT1, p53 and LMP1, we have developed and used various episomal inducible vectors. One of the characteristics of these vectors is that the gene of interest is expressed from a bidirectional doxycycline regulatable promoter that allowing simultaneous expression of truncated NGF receptor, used as a surrogate marker of inducibility. We have subcloned the following cDNAs into these vectors: dominant negative IkBa, active (STAT1a) and dominant inactive natural isoform of STAT1 (STAT1b), p53, a dominant negative mutant of LMP1 (LMP1CT) and LMP1 wild type. After stable transfection of an LCL, induction of the cDNA of interest was performed with doxycycline for 24H. Positive cells for NGFR were purified using magnetic beads and were additionally treated with TNFa, IFNg or Fludarabine for 24H. To control the latency III program, we have used the EREB2-5 cell line harboring an estrogen-regulatable EBNA2 gene. The function of CD95 was studied by quantifying apoptosis induction after CD95 cross linking with a specific antibody. We found that induction of latency III program in EREB 2-5 cells was associated with an increase of both p53 and CD95 expression. Inhibition of LMP1 by LMP1CT decreased the expression of CD95. CD95 expression was hardly regulated by p53. Inhibition of both NF-kB and STAT1 decreased CD95 expression. Only IFNg was able to increase expression of CD95. Finally, we show that induction of CD95 expression by EBV render the infected B-cell sensitive to the induction of CD95-mediated apoptosis. Our results suggest that the latencyc III program of EBV sensitize the infected B-cells to CD95-mediated apoptosis via STAT1 and NF-kB activated by LMP1. In the view of the virus/host equilibrium, this process may render EBV-infected B-cells in latency III susceptible to elimination by the immune system.
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De Curtis, Susan, and Stephen Aderaye. "Additive effect of TGF-ß with TNF-α on type VII collagen gene expression to activate the expression of an extracellular matrix-related gene." American Journal of BioMedicine 2, no. 2 (June 2, 2014): 106–15. http://dx.doi.org/10.18081/2333-5106/014-01/56-66.
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Type VII collagen is the predominant, if not the exclusive, component of the anchoring fibrils, attachment structures stabilizing the association of the cutaneous basement membrane to the underlying dermis. In the skin, type VII collagen is synthesized by both dermal fibroblasts and epidermal keratinocytes. Alterations in the type VII collagen protein structure or lack of its expression due to mutations in the corresponding gene COL7A1 are the hallmark of dystrophic epidermolysis bullosa, a mechano-bullous skin disease characterized by extreme fragility of the skin and leading to development of sub-lamina densa blisters. In this study, we have examined whether the additive effect of TGF-ß with TNF-α on type VII collagen gene expression is exerted at the transcriptional level by activation of the corresponding a promoter. Specifically, we demonstrate that the TNF-α effect is mediated by NF-kB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kB complexes binding the TNF-α response element (TaRE) located in the region [7252/7230], with RelA acting as the transcriptional activator. We provide definitive evidence for the role of both TGF-ß and TNF-α response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-ß and TNF-α. This study provides the functional interaction between the two immediate-early transcription factors, SMAD and NF-kB, to activate the expression of an extracellular matrix-related gene, COL7A1.
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Rahayu, Masruroh, M. Aris Widodo, Diana Lyrawati, and Edi Widjadjanto. "ALPHA-PINENE ATTENUATES MICROGLIAL NF-ΚB ACTIVATION AND INOS EXPRESSION IN GP120-INDUCED NEUROINFLAMMATION." MNJ (Malang Neurology Journal) 7, no. 1 (January 1, 2021): 80–84. http://dx.doi.org/10.21776/ub.mnj.2020.007.01.16.
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Background: Neuro-inflammation plays a role in the pathogenesis of HIV-associated dementia (HIV). Activation of microglia is essential for triggering inflammatory-mediated neurotoxicity. HIV-1 120 kDa envelope glycoprotein (gp120) induces microglial NF-κB signaling which in turn induce pro-inflammatory and iNOS gene transcription. Continuous or excessive activation of NF-κB signaling lead to persistent production of TNF-α and nitric oxide by microglia and induce neuronal apoptosis. Alpha-pinene is a natural substance found in pine tree and has efficacy on inhibiting NF-κB signaling. Objective: This study was designed as a true experimental study and aimed to investigate the effect of alpha-pinene administration toward inflammatory response represented by the percentage of microglia containing activated NF-κB and iNOS expression. Methods: Neuron-glia primary culture from brain tissue of rat fetus was divided into 5 groups as follows: negative control; positive control (gp120 1nM); treatment I, II, and III (gp120 1 nM + alpha-pinene 0.4 µg/mL, 2 µg/mL, and 10 µg/mL, respectively). Microglial NF-κB and iNOS expression were analyzed using immunohistochemistry method. Neuronal apoptosis was measured by TUNNEL method. Results: Result showed that alpha-pinene administration on gp120-treated neuron-glia at all dosages decrease NF-kB activation, iNOS expression, and apoptotic neuron significantly as compared to the gp120-only treated group (p<0.05). Furthermore, alpha-pinene did not affect NF-kB activation and neuronal apoptosis (p>0.05), but significantly elevate iNOS expression (p<0.05) mainly in dosage I and II. Conclusion: We concluded that alpha-pinene has neuroprotective effect on gp120-treated neuron-glia cells through modulation of NF-kB and iNOS expression thus inhibit neuronal apoptosis. Keywords: apoptosis, gp120, iNOS, neuroinflammation, NF-kB
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Ishihara, Yasuhiro, Sarah Y. Kado, Christiane Hoeper, Shelly Harel, and Christoph F. A. Vogel. "Role of NF-kB RelB in Aryl Hydrocarbon Receptor-Mediated Ligand Specific Effects." International Journal of Molecular Sciences 20, no. 11 (May 30, 2019): 2652. http://dx.doi.org/10.3390/ijms20112652.
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Here, we investigate the role of RelB in the regulation of genes which were identified to be induced in an aryl hydrocarbon receptor (AhR)-dependent manner and critically involved in regulation of immune responses. We analyzed the expression of genes of the AhR gene battery, cytokines, and immune regulatory enzymes in bone marrow-derived macrophages (BMM) and thymus of B6 wildtype (wt) mice and RelB knockout (RelB−/−) mice after treatment with various AhR ligands. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced expression of indoleamine 2,3-dioxygenase 1 (IDO1) and IDO2 was significantly repressed in thymus of RelB−/− mice but not in BMM derived from RelB−/− mice. Interestingly, the induced and basal expression of the cytokines interleukin (IL)-17A, IL-22, and CCL20 required the functional expression of RelB. The RelB-dependent expression of CCL20 was induced by the AhR ligands TCDD and 6-formylindolo[3,2-b]carbazole (FICZ), whereas indole-3-carbinol (I3C) suppressed CCL20 in lipopolysaccharide (LPS)-activated wt BMM. The LPS-induced expression of IL-6 and IL-10 was enhanced by TCDD and FICZ, whereas I3C significantly suppressed these cytokines in BMM. The exposure to FICZ led to higher increases of IL-17A and IL-22 mRNA compared to the effect of TCDD or I3C in thymus of wt mice. On the other hand, TCDD was the strongest inducer of CYP1A1, AhR Repressor (AhRR), and IDO2. In summary, these findings provide evidence for the important role of RelB in the transcriptional regulation of cytokines and enzymes induced by AhR ligands.
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Asare, Yaw, Erdenechimeg Shagdarsuren, Johannes Schmid, Pathricia Tilstam, Jochen Grommes, Omar El Bounkari, Anke Schütz, et al. "Endothelial CSN5 impairs NF-κB activation and monocyte adhesion to endothelial cells and is highly expressed in human atherosclerotic lesions." Thrombosis and Haemostasis 110, no. 07 (2013): 141–52. http://dx.doi.org/10.1160/th13-02-0155.
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SummaryThe COP9 signalosome (CSN), a multifunctional protein complex involved in the regulation of cullin-RING-E3 ubiquitin ligases (CRLs), has emerged as a regulator of NF-κB signalling. As NF-κB drives the expression of pro-inflammatory and pro-atherosclerotic genes, we probed the yet unknown role of the CSN, in particular CSN5, on NF-KB-mediated atherogenic responses in endothelial cells. Co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs) revealed the presence of a super-complex between IKK and CSN, which dissociates upon TNF-α stimulation. Furthermore, CSN5 silencing enhanced TNF-α-induced IKB-α degradation and NF-κB activity in luci-ferase reporter assays. This was paralleled by an increased NF-KB-driven upregulation of atherogenic chemokines and adhesion molecules, as measured by qPCR and flow cytometry, and translated into an enhanced arrest of THP-1 monocytes on TNF-α-stimulated, CSN5-depleted HUVECs. Reverse effects on NF-κB activity and THP-1 arrest were seen upon CSN5 overexpression. Finally, double-immunostaining confirmed the expression of CSN subunits in the endothelium of human atherosclerotic lesions, and revealed an increased expression of CSN5 which correlated with atheroprogression. In conclusion, endothelial CSN5 attenuates NF-KB-dependent pro-inflammatory gene expression and monocyte arrest on stimulated endothelial cells in vitro, suggesting that CSN5 might serve as a negative regulator of atherogenesis.Note: The review process for this manuscript was fully handled by G. Y. H. Lip, Editor in Chief.
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Kagoya, Yuki, Akihide Yoshimi, Shunya Arai, Keisuke Kataoka, Masahiro Nakagawa, Keiki Kumano, and Mineo Kurokawa. "NF-κB/TNF-α Positive Feedback Loop with Active Proteasome Machinery Supports Myeloid Leukemia Initiating Cell Capacity." Blood 120, no. 21 (November 16, 2012): 654. http://dx.doi.org/10.1182/blood.v120.21.654.654.
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Abstract Abstract 654 Acute myeloid leukemia (AML) is an aggressive hematologic malignancy arising from leukemia initiating cells (LIC), and is comprised of highly heterogeneous groups with different cytogenetic and molecular abnormalities, which makes it difficult to establish a broadly effective therapeutic strategy. Since constitutive NF-kB pathway activation has been reported in different types of AML cells, it is one of the promising candidates which are universally involved in the LIC phenotype. However, the mechanism of activation and its significance in leukemia progression have not been studied well. In this study, we explored NF-kB pathway activity and its role in LICs using various myeloid leukemia mouse models including MLL-ENL, MOZ-TIF2, and BCR-ABL/Nup98-HoxA9 leukemias. A number of NF-kB target genes showed elevated expression in LIC of each model: leukemic granulocyte-monocyte progenitors (L-GMP) in MLL-ENL or MOZ-TIF2 model and lineage- Sca-1+ fraction in BCR-ABL/Nup98-HoxA9 model, compared with normal hematopoietic stem cells (HSC) and GMP. Moreover, in immunofluorescence staining, each type of LIC displayed prominent nuclear translocation of NF-kB subunit p65. On the other hand, p65 was localized mainly in the cytoplasm in normal cells and, interestingly, non-LIC fraction in the bone marrow cells of the leukemia mice. To our surprise, LIC retained NF-kB activity even after serum-free culture, indicating that NF-kB pathway is prevalently activated in LIC in an autonomous fashion. To study the mechanism of activation, we analyzed gene expression profiles of LIC and normal HSC in murine and human AMLs and found that LIC showed distinctly elevated expression of TNF-a, one of the major activators of the NF-kB pathway. Consistent with these results, the culture media conditioned by LIC had higher TNF-a levels than those of normal cells. In all of the three types of leukemia mice, bone marrow extracellular fluid included higher TNF-a than that of control mice. Importantly, TNF-a blockage by the neutralizing antibody significantly attenuated p65 nuclear localization in LIC, and NF-kB inhibition by expression of a super repressor form of IkBa (IkB-SR) suppressed the TNF-a expression in LIC, indicating that LIC maintains its NF-kB activity by autocrine NF-kB/TNF-a positive feedback loop. Disruption of this loop by induction of IkB-SR or shRNA-mediated knockdown of TNF-a significantly reduced colony-forming abilities of leukemia cells and prolonged survival of leukemic mice in all the three models. In contrast, transduction of IkB-SR into normal HSC exerted no influence on their colony-forming ability. These results suggested that the NF-kB/TNF-a positive feedback loop plays a vital role for LIC propagation. We also addressed the mechanism of the difference in NF-kB activity between LIC and non-LIC. Notably, LIC had decreased protein levels of IkBa compared with non-LIC in spite of the same mRNA expression levels between them. In addition, basal 20S proteasome activity and the expression levels of proteasome subunit genes in LIC were higher than those in non-LIC, indicating that enhanced proteasomal degradation of IkBa could lead to selectively high NF-kB activity of LIC. The same propensity was seen in human AML CD34+CD38- cells versus CD34- cells in the analysis of microarray expression data. Proteasome inhibition by bortezomib diminished the differences of IkBa protein level. Moreover, its administration to leukemic mice selectively killed LIC fraction and prolonged survival in the in vivo transplantation model. Finally, forcible maintenance of NF-kB activity in LIC by shRNA-mediated knockdown of IkBa significantly enhanced its self-renewal activity as determined by surface marker profiles after in vitro culture. In the analysis of mice reconstituted with the IkBa-downregulated leukemic cells, bone marrow mononuclear cells had increased colony-forming cell ability and enhanced LIC frequency as determined by in vivo limiting dilution serial transplantation assay. These results indicated that the transition from LIC to non-LIC might be associated with the attenuation of NF-kB activity due to inefficient degradation of IkBa. In summary, these findings elucidate that NF-kB/TNF-a signaling in LIC, under support of the proteasome activity, has a critical role for both maintenance and propagation of LIC and provide a widely applicable approach for targeting LIC in myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.
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Amin, Jakia, Ken-ichiro Otsuyama, Emi Uchida, Saeid Abroun, Karim Shamsasenjan, Khademul Islam, and Michio M. Kawano. "The Potentials of Constitutive NF-κB Activation in U266 Cell Lines." Blood 108, no. 11 (November 16, 2006): 5008. http://dx.doi.org/10.1182/blood.v108.11.5008.5008.
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Abstract NF-κB has been described as a family of ubiquitously expressed transcription factors, composed of a dimer of distinct members of the Rel protein family (RelA/p65, cRel, RelB, p50, p52), and after stimulation, NF-κB dimer translocate into the nucleus to activate the transcription of various target genes. In the present study, we aimed to elucidate the mechanism of constitutive activation of NF-κB in human myeloma cells. For this purpose here, we used a human myeloma cell line, U266 cells, and performed gel shift assay and also examined the expression of different target genes of NF-κB by RT-PCR and determined the up-regulation of some of the anti-apoptopic genes (IAP-1, IAP-2, Survivin, Bcl-2, Bfl-1) and cell cycle regulators (Cox-2, Cyclin-D1, Cyclin-D2). This NF-κB-mediated gene expression provided an approach for investigating the protein expression of NF-κB family in U266. Our data revealed that the expression of p65, p50, RelB and cRel was strongly detected in the whole lysate of U266, while weak expression of p52 was found in U266. Furthermore, in the nuclear fractions of U266 cells, we strongly detected the expression of p65, RelB and p50, but not the cRel, for cRel was present in the cytoplasm. It has been recognized that dimerization is essential for NF-κB activity. Then, we performed immunoprecipitation-immunoblot analysis to demonstrate the dimer that might present in the U266, and we detected the possible dimerization such as p65-RelB in the nucleus. Therefore, these results suggest that U266 shows high constitutive activation of NF-kB, and possible hetero- and homodimer of NF-kB except for p52 may be responsible for the constitutive activation of NF-kB in U266 cells.
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Milone, Michael C., Roddy OConnor, Michael May, Steven Albelda, and Benjamin Philipson. "4-1BB-Costimulated CAR-Mediated Non-Canonical NF-Kb Signaling Enhances CAR T Cell Survival and Suppresses Bim Expression." Blood 132, Supplement 1 (November 29, 2018): 3713. http://dx.doi.org/10.1182/blood-2018-99-119522.
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Abstract Chimeric Antigen Receptor (CAR) T cell therapy induces deep and durable responses in a large percentage of patients with B-cell malignancies. These responses often correlate with CAR T cell persistence in patients. The first two FDA-approved CAR-T cell therapies employ 2nd generation CARs that use different costimulatory domains derived from either CD28 or 4-1BB. Data from pre-clinical studies as well as clinical trials suggest that 4-1BB CAR (BBz) T cells persist longer than CD28 CAR (28z) T cells. One signal associated with cellular survival and activated by endogenous 4-1BB, but not CD28, is the non-canonical NF-kB (ncNF-kB) pathway. NcNF-kB is required for memory T cell persistence and enhances tumor cell survival by suppressing pro-apoptotic gene transcription. Therefore, we hypothesize that the BBz, but not the 28z CAR, activates ncNF-kB signaling, which promotes CAR T cell persistence by suppressing pro-apoptotic gene expression. Using primary human T cells isolated from the apheresis products of anonymous healthy donors, we generated 28z or BBz T cells by lentiviral transduction following activation by CD3 and CD28. Induction of ncNF-kB signaling following CAR activation was assessed by western blot. NcNF-kB function was evaluated by concomitant expression of a dominant-negative mutant of NF-kB-inducing Kinase (dnNIK) to block the ncNF-kB pathway. In addition to pathway analysis by western blotting, T cell proliferation, immunophenotype and survival were assessed by bead-based counting via flow cytometry. To explore mechanisms affecting survival, pro- and anti-apoptotic gene expression was analyzed by qPCR and western blot over three weeks following restimulation through the CAR. Following CAR activation, ncNF-kB signaling was detected in BBz but not 28z T cell lysates. Control mRFP BBz T cells expanded approximately 10 fold more than BBz T cells coexpressing dnNIK. DnNIK-expressing BBz T cells exhibited higher rates of cell death contributing to the observed differences apparent in ex vivo expansion. This cell death was associated with a 2 fold increase in the message and nearly 3 fold increase in the expression of the pro-apoptotic protein, Bim, in the dnNIK BBz T cells relative to control. In contrast, dnNIK 28z T cells had no appreciable difference in proliferation or survival compared with control. Therefore, BBz, but not 28z signaling activates the ncNF-kB pathway, which protects BBz T cells from cell death likely by restricting the expression of the pro-apoptotic protein, Bim. Disclosures Milone: Novartis: Patents & Royalties.
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Gu, Lubing, Ningxi Zhu, Harry W. Findley, William G. Woods, and Muxiang Zhou. "Identification and Characterization of the IKKα Promoter." Journal of Biological Chemistry 279, no. 50 (October 5, 2004): 52141–49. http://dx.doi.org/10.1074/jbc.m407915200.
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IKKα, a subunit of IkBα kinase (IKK) complex, has an important role in the activation of nuclear factor-kB (NF-kB), a key regulator of normal and tumor cell proliferation, apoptosis, and response to chemotherapy. However, little is known about the transcriptional regulation of theIKKα gene itself. The present study revealed that the transcriptional induction of theIKKα gene is positively regulated by binding ETS-1, the protein product of theETS-1proto-oncogene. Furthermore, ETS-1 mediated activation of IKKα is negatively regulated by p53 binding to ETS-1. By analyzing the genomic DNA sequence, we identified the putativeIKKα promoter sequence in the 5′-flanking untranslated region of theIKKα gene. Transfection of EU-4, an acute lymphoblastic leukemia (ALL) cell line, with plasmids containing theIKKα 5′-untranslated region sequence upstream of the luciferase reporter showed that this region possessed major promoter activity. Induction or enforced overexpression of p53 represses IKKα mRNA and protein expression as well asIKKα promoter activity. Deletion and mutation analyses as well as chromatin immunoprecipitation and electrophoretic mobility shift assay indicated that ETS-1 binds to the coreIKKα promoter and strongly induces its activity. Although p53 does not directly bind to theIKKα promoter, it physically interacts with ETS-1 and specifically inhibits ETS-1-inducedIKKα promoter activity. These results suggest that the proximal 5′-flanking region of theIKKα gene contains a functional promoter reciprocally regulated by p53 and ETS-1. Furthermore, loss of p53-mediated control over ETS-1-dependent transactivation ofIKKα may represent a novel pathway for the constitutive activation of NF-kB-mediated gene expression and therapy resistance in cancer.
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Otsuyama, Ken-ichiro, Zi Ma, Shangqin Liu, Saeid Abroun, Hideki Asaoku, and Michio M. Kawano. "PPARβ-Mediated Suppression of the Growth and Survival in Human Myeloma Cells Conteracting NF-kB Activity." Blood 106, no. 11 (November 16, 2005): 5053. http://dx.doi.org/10.1182/blood.v106.11.5053.5053.
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Abstract PPAR(peroxisomal proliferators-activated receptor)β is considered to be involved in the lipid metabolism and regulating the inflammatory response and in human myeloma cells PPARβis predominantly expressed among these PPARs. However, it remains to be clarified what is the functional role of PPARβ in myeloma cells. In order to identify what are the ligands for PPARβ, we constructed the PPREβ-luciferase reporter gene and performed the reporter assay in Hela cells. Since we have already identified several reagents (DHEA (dehydroepiandrosterone), DHEA-S, baicalein, baicalin, dexamethasone) that suppressed the growth or survival of human myeloma cells (Cancer Res65:2269,2005; Blood105:3312,2005), we analyzed whether these reagents augmented the expression of reporter gene. DHEA-S, baicalein and dexamethasone showed the possibility of their PPARβ binding. Furthermore, these reagents could induce or upregulate the expression of PPREβ-target genes such as ILK and COX2 in myeloma cell lines. We’ve already confirmed that DHEA-S inhibited the proliferation and survival of primary myeloma cells as well as myeloma cell lines, and downregulated the activity of NF-kB, also baicalein showed the growth suppression through the downregulation of NF-kB. Therefore, it is possible that PPARβ-binding reagents such as DHEA-S and baicalein could counteract NF-kB activity to suppress the growth or survival in myeloma cells, while the exact mechanism is under investigation.
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Li, Ailing, Ying Liang, Yi Liu, and Gary VanZant. "Latexin Regulates Stem Cell Numbers Via a NF-êB-Dependent Pathway." Blood 112, no. 11 (November 16, 2008): 1400. http://dx.doi.org/10.1182/blood.v112.11.1400.1400.
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Abstract Latexin was first discovered in the developing lateral cortex of the brain and is the only known endogenous carboxypeptidase A (CPA) inhibitor in mammals. Our recent studies have revealed that latexin exerted a novel function in regulating murine hematopoietic stem cell number by repressing stem cell self-renewal and enhancing apoptosis (Liang et al., Nat. Gen.39:178, 2007) . However, the underlying mechanism remains obscure. To test whether latexin acts through its canonical mechanism in hematopoietic cells we used fluorescence immunohistochemistry to reveal that latex and CPA did not co-localize intracellularly. Moreover, in contrast with latexin, an unrelated inhibitor of CPA did not affect hematopoietic stem cell numbers in vivo. Thus, latexin appeared to act through a novel pathway unrelated to the inhibition of CPA. To identify novel intracellular protein partners of latexin, tandem affinity purification (TAP) tag-labeled latexin was overexpressed in FDCP1, a primitive hematopoietic progenitor cell line. Latexin-interacting proteins were isolated from lysates of FDCP1-TAP-latexin and FDCP-1-TAP control by TAP. Purified eluates were screened by mass spectrometry proteomic technologies. Eleven proteins, but not CPA, were identified as latexin-interacting partners by LC-MS analysis. Using Western blots and co-immunoprecipitation we found that latexin interacted with a novel binding partner, ribosome protein S3 (RPS3), a moiety recently found to complex with NF-kB and thus regulate its selective activation of target genes (Wan et al. Cell131:927, 2007). We hypothesized that latexin may compete with NF-kB for binding RPS3 and thus indirectly affect NF-kB-directed gene expression. Thus, in order to determine if NF-kB-responsive genes were differentially regulated in primitive hematopoietic cells in response to latexin modulation, we made use of a mouse model congenic for the latexin locus. We introgressed the high-expressing C57BL/6 latexin allele onto the normally low-expressing DBA/2 genetic background. We then compared the gene expression profiles of Lineage negative, Sca-1 positive, and c-kit positive (LSK) cells isolated from the bone marrow of congenic and DBA/2 background (control) mice using microarrays. A functional cluster of genes known to be regulated proximally by NF-kB and which are related to mechanisms of latexin action--namely apoptosis and cell replication—were differentially expressed in concordance with the latexin allele present. These results are consistent with a model in which latexin impeded RPS3-NF-kB complex formation and subsequently altered NF-kB-mediated gene transcription. Taken together, our findings provide a potential mechanism by which latexin regulates hematopoietic stem cell number via a heretofore unappreciated pathway. Liang Y, M Jensen, B Aronow, H Geiger, G Van Zant. The quantitative trait gene latexin influences the size of the hematopoietic stem cell population in mice. Nature Genetics 39:178–88, 2007. Wan F, DE Anderson, RA Bamitz, A Snow, N Bidere, L Zheng, V Hegde, LT Lam, LM Staudt, D Levens, WA Deutsch, MJ Lenardo. Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation. Cell 131:927–39, 2007.
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Kammala, Ananth Kumar, Samantha Sheller-Miller, Enkhtuya Radnaa, Talar Kechichian, Hariharan Subramanian, and Ramkumar Menon. "Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation." International Journal of Molecular Sciences 21, no. 20 (October 20, 2020): 7747. http://dx.doi.org/10.3390/ijms21207747.
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The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.
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WILD, Angela C., Jerry J. GIPP, and R. Timothy MULCAHY. "Overlapping antioxidant response element and PMA responseelement sequences mediate basal and β-naphthoflavone-induced expression of the human γ-glutamylcysteine synthetase catalytic subunit gene." Biochemical Journal 332, no. 2 (June 1, 1998): 373–81. http://dx.doi.org/10.1042/bj3320373.
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γ-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in the de novo synthesis of GSH, is a heterodimer, consisting of a catalytic (GCSh) and a regulatory subunit (GCSl). We previously demonstrated that the constitutive and β-naphthoflavone (β-NF)-induced expression of the GCSh gene is mediated by a distal antioxidant response element (ARE), ARE4, located 3.1 kb upstream of the transcriptional start site [Mulcahy, Wartman, Bailey and Gipp (1997) J. Biol. Chem. 272, 7445–7454]. ARE4 consists of a consensus ARE sequence (5´-GTGACTCAGCG-3´) containing an embedded PMA-responsive element (TRE, underlined). The relative significance of the two overlapping response elements to constitutive and β-NF-induced expression of the GCSh gene was determined by mutational analyses. The internal activator protein-1 (AP-1)-binding sequence mediated constitutive expression of promoter/reporter transgenes, but was not required for β-NF responsiveness. In gel-shift experiments, the TRE was necessary for binding of proteins from nuclear extracts prepared from untreated HepG2 cells. In contrast, induction by β-NF was dependent on an intact ARE sequence, particularly the terminal GC box of ARE4. The GC box of ARE4 was shown to be essential for both basal and β-NF-induced expression of reporter constructs. This element also influenced binding of nuclear proteins to ARE4, specifically in extracts isolated from β-NF-treated HepG2 cells. Because previous studies indicated that ARE4 may co-operate with a separate putative ARE, the role of the neighbouring sequence (ARE3), located 34 bases downstream of ARE4, was also evaluated. Mutation of this element within a GCSh promoter/reporter did not modify the basal or β-NF-induced expression of the transgene, demonstrating that ARE3 does not influence the constitutive or β-NF-induced expression of the GCSh gene.
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Imamura, Yasuhiro, Yoshimasa Makita, Kazuya Masuno, and Hourei Oh. "Inhibitory Mechanism of IL-6 Production by Orento in Oral Squamous Cell Carcinoma Cell Line CAL27 Stimulated by Pathogen-Associated Molecular Patterns from Periodontopathogenic Porphyromonas gingivalis." International Journal of Molecular Sciences 24, no. 1 (December 31, 2022): 697. http://dx.doi.org/10.3390/ijms24010697.
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Orento is a traditional Japanese medicinal kampo preparation that is also prescribed in oral care. In oral squamous cell carcinoma cell line CAL27, orento significantly inhibited periodontopathogenic bacterium Porphyromonas gingivalis lipopolysaccharide (LPS) and lipoproteins (PAMP)-stimulated production of interleukin (IL)-6. This suggests that orento negatively regulates PAMP-mediated toll-like receptor (TLR) signaling. Orento significantly suppressed PAMP-stimulated activation of the IL-6 promoter, indicating that orento may suppress the production of IL-6 by PAMP at the transcriptional level. Orento also suppressed TLR-mediated activation of transcription factor nuclear factor-kappa B (NF-kB) that was stimulated by PAMP. This finding indicates that orento may suppress the function and activation of factors involved in TLR signaling, thereby suppressing NF-kB-dependent expression of various genes. Orento suppressed IL-1 receptor-associated kinase (IRAK4), IRAK1, and c-Jun N-terminal kinase (JNK) phosphorylation in PAMP-stimulated CAL27 cells. This result indicates that orento is involved in the initiation of TLR signaling by PAMP and suppresses the downstream signaling pathways of myeloid differentiation primary response gene 88 (MyD88) such as mitogen-activated protein kinase (MAPK) and NF-kB cascades. These findings suggest that orento has an inhibitory effect on the production of inflammatory cytokines.
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Park, JH, and L. Levitt. "Overexpression of mitogen-activated protein kinase (ERK1) enhances T- cell cytokine gene expression: role of AP1, NF-AT, and NF-KB." Blood 82, no. 8 (October 15, 1993): 2470–77. http://dx.doi.org/10.1182/blood.v82.8.2470.2470.
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Abstract Transfected Jurkat cells overexpressing extracellular signal-regulated kinase (ERK1), also referred to as mitogen-activated protein (MAP) kinase, were selected by Western blotting assay using anti-ERK1 and antiphosphotyrosine antibodies in combination with a functional MAP kinase assay. We then asked whether enhanced ERK1 expression had any effect on induction of T-cell cytokine genes. The results show that overexpression of ERK1 enhances expression of T-cell interleukin-2 (IL- 2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA; no change was seen in expression of the alpha-actin gene. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in ERK1-overexpressing clones relative to nontransformed or vector-transformed cells, whereas no enhancement of CK1-CK2 protein DNA binding activity was detected after ERK1 overexpression. Additionally, increased NF-AT DNA binding activity was associated with functional enhancement of NF-AT transactivating activity in ERK1-overexpressing cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokine gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.
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Park, JH, and L. Levitt. "Overexpression of mitogen-activated protein kinase (ERK1) enhances T- cell cytokine gene expression: role of AP1, NF-AT, and NF-KB." Blood 82, no. 8 (October 15, 1993): 2470–77. http://dx.doi.org/10.1182/blood.v82.8.2470.bloodjournal8282470.
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Transfected Jurkat cells overexpressing extracellular signal-regulated kinase (ERK1), also referred to as mitogen-activated protein (MAP) kinase, were selected by Western blotting assay using anti-ERK1 and antiphosphotyrosine antibodies in combination with a functional MAP kinase assay. We then asked whether enhanced ERK1 expression had any effect on induction of T-cell cytokine genes. The results show that overexpression of ERK1 enhances expression of T-cell interleukin-2 (IL- 2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA; no change was seen in expression of the alpha-actin gene. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in ERK1-overexpressing clones relative to nontransformed or vector-transformed cells, whereas no enhancement of CK1-CK2 protein DNA binding activity was detected after ERK1 overexpression. Additionally, increased NF-AT DNA binding activity was associated with functional enhancement of NF-AT transactivating activity in ERK1-overexpressing cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokine gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.
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Arai, Ayako, Mayumi Takahashi, Ken-Ichi Imadome, Takatoshi Koyama, Yasunori Saitoh, Shoji Yamaoka, Shigeyoshi Fujiwara, and Osamu Miura. "NF-κB Is Constitutively Activated in EBV-Infected T or NK Cells and Can Be a Molecular Target for EBV-Positive T/NK-Cell Lymphoproliferative Disease Treatment." Blood 116, no. 21 (November 19, 2010): 1998. http://dx.doi.org/10.1182/blood.v116.21.1998.1998.
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Abstract Abstract 1998 Epstein-Barr virus (EBV) can infect not only B cells but also rarely T or NK cells and causes EBV-positive T/NK-cell lymphoproliferative disease (EBV-T/NK-LPD), such as extranodal NK/T-cell lymphoma (ENKL), aggressive NK-cell leukemia, and chronic active EBV infection (CAEBV). However, why and how EBV infects T or NK cells and the mechanism of action responsible for the development of these EBV-induced malignancies have not been elucidated to date. Furthermore, optimal chemotherapy for these, especially for CAEBV, has not been established and the prognosis of EBV-T/NK-LPD remains very poor. NF-kB is a transcription factor that mediates anti-apoptotic molecular signaling and promotes proliferation of cancer cells. NF-kB is known to be constitutively activated in some hematological malignancies; the proteasome inhibitor bortezomib suppresses its activity and is used clinically as an anticancer reagent. To clarify the molecular mechanism underlying the development of EBV-T/NK-LPD, we focused on NF-kB in four EBV-positive T- and NK-cell lines, SNT8, SNT15, SNT16, and SNK6, which had been established from primary lesions of ENKL patients (SNT8 and SNK6) and the peripheral blood (PB) of a CAEBV patient (SNT15, 16), respectively. In these cells, it was demonstrated that p50, p52, RelA, and RelB—the components of NF-kB—existed constitutively in the nucleus. On the other hand, the EBV-negative T-cell lines, Jurkat and Molt4, and an NK-cell line, KHYG1, were negative for nuclear localization of these molecules. Elevated nuclear NF- kB–DNA binding activity was demonstrated by electrophoretic mobility shift assay (EMSA), using oligonucleotides encoding an NF-kB-binding sequence as a probe. Supershift EMSA revealed that NF- kB–DNA binding complexes in these cells involved p50, p52, and RelA. In addition, reporter assay using NF-kB-dependent luciferase reporter gene consisting of an NF-kB-binding site as a promoter demonstrated constitutive activation of NF-kB in SNT8 cells, whereas not in Jurkat cells. From these results, we concluded that NF-kB was constitutively activated through the canonical and noncanonical pathways in these EBV-infected T- or NK-cell (EBV-T/NK cell) lines. Next, we investigated NF-kB activation in EBV-T/NK cells derived from CAEBV patients. CAEBV was diagnosed according to the following criteria: presence of persistent infectious mononucleosis-like symptoms, elevation of EBV-DNA titer in PB, and detection of EBV-infected T or NK cells with clonal proliferation. To detect infected cells, we isolated peripheral mononuclear cells and divided them into CD19-, CD4-, CD8-, or CD56-positive fractions using antibody-conjugated magnetic beads. The EBV DNA of each fraction was quantified using a real-time quantitative polymerase chain reaction assay. Ten patients (aged 8–72 years; 4 male, 6 female; infected cell types CD4:3, CD8:3, γδ:1, and CD56:3) were diagnosed as CAEBV. Then, we examined NF-kB activation in these cells. Similar to the cell lines, immunoblotting and EMSA demonstrated evidence of constitutive activation of NF-kB such as nuclear localization and DNA binding of p50, p52, and RelA in EBV-T/NK cells from CAEBV patients. To investigate the molecular mechanism of NF-kB activation in EBV-T/NK cells, we examined the influence of viral proteins expressed in EBV-T/NK cells, including LMP1, LMP2A, LMP2B, and EBNA1 (latency type 2). We performed luciferase reporter assay using expression vectors for these proteins in Jurkat cells. LMP1, most significantly, and LMP2A, to a lesser extent, upregulated NF-kB-dependent reporter gene expression in Jurkat cells, whereas the other viral proteins did not, suggesting that LMP1 and LMP2A mediated NF-kB activation in T/NK cells. Finally we investigated the contribution of NF-kB to the development of EBV-T/NK-LPD. Bortezomib, in a dose-dependent manner, inhibited translocation of p50, p52, and RelA to the nucleus and reduced the expression of Bcl-XL, which was a transcriptional target of NF-kB in SNT8, SNK6, and CAEBV patients’ cells. In addition, it was also confirmed that bortezomib (0.5nM -5nM) suppressed the survival of and induced apoptosis of EBV-T/NK cells derived from 5 CAEBV patients. These results suggest that constitutive activation of NF-kB contributes to the development of EBV-T/NK-LPD and that the NF-kB signaling pathway can be an attractive molecular target for treatment. Disclosures: No relevant conflicts of interest to declare.
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Suh, Han Na, Young Kyu Kim, Ju Young Lee, Goo-Hwa Kang, and Jeong Ho Hwang. "Dissect the immunity using cytokine profiling and NF-kB target gene analysis in systemic inflammatory minipig model." PLOS ONE 16, no. 6 (June 4, 2021): e0252947. http://dx.doi.org/10.1371/journal.pone.0252947.
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Minipigs have remarkably similar physiology to humans, therefore, they it can be a good animal model for inflammation study. Thus, the conventional (serum chemistry, histopathology) and novel analytic tools [immune cell identification in tissue, cytokine level in peripheral blood mononuclear cells (PBMC) and serum, NF-kB target gene analysis in tissue] were applied to determine inflammation in Chicago Miniature Swine (CMS) minipig. Lipopolysaccharide (LPS)-induced acute systemic inflammation caused liver and kidney damage in serum chemistry and histopathology. Immunohistochemistry (IHC) also showed an increase of immune cell distribution in spleen and lung during inflammation. Moreover, NF-kB-target gene expression was upregulated in lung and kidney in acute inflammation and in heart, liver, and intestine in chronic inflammation. Cytokine mRNA was elevated in PBMC under acute inflammation along with elevated absolute cytokine levels in serum. Overall, LPS-mediated systemic inflammation affects the various organs, and can be detected by IHC of immune cells, gene analysis in PBMC, and measuring the absolute cytokine in serum along with conventional inflammation analytic tools.
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Molinero, Luciana, Michelle Miller, Mona Mashayekhi, Luqiu Chen, Ping Zhou, Nicole Tramontini, Monica Michelotti, Xindong Liu, Xiaoping Zhu, and Maria-Luisa Alegre. "NF-κB controls IL-7 receptor expression in T cells (P6295)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 184.9. http://dx.doi.org/10.4049/jimmunol.190.supp.184.9.
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Abstract IL-7 and tonic TCR signaling control naïve T cell homeostasis. The transcription factor NF-κB plays a key role during T cell activation, proliferation and survival, but little is known on the role of basal NF-κB in quiescent T cells. Using mice bearing NF-κB impaired T cells (IκBαΔN), we show that basal levels of NF-κB signaling in naïve T cells are required for cell survival both in vivo and in vitro. In vitro, IκBαΔN T cells display reduced survival to IL-7, IL-7-mediated STAT5 phosphorylation and Bcl2 upregulation. Indeed, protein levels of IL-7Rα, but not common γc subunit (CD132), are markedly reduced in IκBαΔN naïve T cells. Assessing mice deficient for NF-κB1 and NF-κB2 subunits, canonical (NF-κB1) but not alternate (NF-κB2) NF-κB pathway is required for IL7Rα expression in T cells. Conversely, constitutive activation of NF-κB in T cells (Lck-Cre x IKKβCA) enhances IL-7Rα expression and IL-7 induced T cell survival. RelA/p65 and NF-κB1/p50 might control transcription through a κB binding region located 3.5 kb upstream of il7r gene, as assessed by EMSA and chromatin immunoprecipitation. Notably, using a NF-κB pharmacological inhibitor we show that NF-κB also controls IL-7Rα expression in human T cells. Taken together, our data suggest that NF-κB regulates il7r transcription both in mice and human, therefore affecting homeostasis of naive T cells.
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Ganan-Gomez, Irene, Yue Wei, Hui Yang, Maria Carmen Boyano-Adánez, and Guillermo Garcia-Manero. "Overexpression Of Mir-125a In Bone Marrow CD34+ cells Of Patients With Myelodysplastic Syndrome Is Correlated To a Poor Prognosis and May Contribute To The Pathogenesis Of The Disease Through The Modulation Of NF-Kb Activation and Enhancement Of Differentiation Arrest." Blood 122, no. 21 (November 15, 2013): 5206. http://dx.doi.org/10.1182/blood.v122.21.5206.5206.
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Abstract Myelodysplastic syndromes (MDS) are a group of clonal malignancies characterized by impaired proliferation and differentiation of hematopoietic stem cells and precursors. The involvement of toll-like receptor (TLR)-mediated signalling in the modulation of myeloid differentiation and its participation in the pathogenesis of MDS are well documented (Wei et al 2013). Increased signaling through this pathway leads to the constitutive activation of NF-kB, which regulates the production of cytokines and mediates cell proliferation and apoptosis (Starczynowski 2010). In addition to the expression of proteins involved in inflammation, the TRL pathway also induces the expression of microRNAs (miRNAs) which participate in the fine-tuning of the inflammatory response (Kawai and Akira 2010). miR-125a and miR-125b are known modulators of hematopoiesis (Gerrits et al. 2012) and have been reported to be involved in several lymphoid and myeloid diseases. Little is known about their role in the pathogenesis of MDS. Interestingly, NF-kB-activating ability has been described for both miR-125a/b (Kim et al. 2012), and miR-125b appears to be upregulated by NF-kB within a positive feedback loop (Zhou et al. 2009; Tan et al. 2012). The aim of this work was to analyze the expression of miR-125a/b in MDS CD34+ cells and to study their relationship with the TLR pathway and differentiation. For this purpose, we analyzed the expression of miR-125a/b by qPCR in bone marrow CD34+ cells of 48 MDS patients, compared it with expression in healthy donors and studied the correlation with overall survival. In our study, we included miR-99b, which is clustered with miR-125a in the genome. Levels of TLR pathway components were detected by qPCR and correlated to those of the miRNAs. Activation of NF-kB was determined in Meg-01 and KG1 cells by the luciferase reporter gene assay, using a vector containing NF-kB responsive elements. Differentiation was studied in K562 and MDS-L cells through colony formation assays combined with analyses of the expression of specific markers by qPCR. For these experiments we used miRNA analogs and a miR-125a anti-sense oligonucleotide. Our results showed that miR-125a, but not miR-125b, is strongly overexpressed in MDS patients (∼15-fold of controls; P<0.01) and that miR-125a levels are significantly and negatively correlated to overall survival of MDS patients (P<0.05). Moreover, expression of miR-99b is also directly connected to the progression of the disease (P<0.05). Both miR-125a and miR-99b cooperate in vitro in the activation of NF-kB (P<0.001); however, we observed a negative correlation between miR-99b/miR-125a expression and levels of TLR2, TLR7 and their downstream proteins MyD88 and JMJD3 (P<0.05), suggesting that NF-kB activation by the miRNA cluster occurs in the absence of TLR signaling. Furthermore, we observed a ∼4-fold increase in NF-kB activity after miR-125a inhibition in the presence of a TLR2 agonist (P<0.001), indicating that miR-125a acts as an NF-kB inhibitor upon TLR stimulation. These results show that miR-125a is involved in the fine-tuning of NF-kB activity and that its effects may depend on the status of the TLR pathway. We then investigated the role of miR-125a in hematopoiesis and found that this miRNA contributes to the blockade of differentiation in the cell lines studied. Therefore, miR-125a could be involved in the pathogenesis or progression of MDS through the modulation of NF-kB activity and differentiation arrest. Thus, this miRNA could be a good prognostic marker and is a potential therapeutic target in MDS. Disclosures: No relevant conflicts of interest to declare.
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Wessel, Alex, Amy Hsu, Jevgenia Zilberman-Rudenko, Raphaela Goldbach-Mansky, Richard Siegel, and Eric Hanson. "Inflammatory disease and impaired antiviral immunity due to unbalanced NF-kB and IRF3 activation result from a de novo human NEMO mutation (P1415)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 57.19. http://dx.doi.org/10.4049/jimmunol.190.supp.57.19.
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Abstract The RIG-I-like receptors mediate antiviral signaling through NEMO-dependent activation of transcription factors NF-kB and IRF3, which are recognized for their roles in inflammation and the induction of Type I Interferon, respectively. We identified an individual with increased susceptibility to viral infection and persistent inflammatory disease. Sanger sequencing revealed a de novo synonymous mutation in the gene encoding NEMO that results in an in-frame splicing event and exclusive production of a shortened protein. To understand the role of NEMO in regulating the antiviral response, we stimulated patient and healthy control fibroblasts with RIG-I-like receptor ligand poly(I:C), which mimics viral RNA. Induction of IRF3 response genes IFNB1 and CXCL10 was reduced 35-fold and 500-fold (n=2), respectively, compared to healthy control, and patient cells were unable to control viral infection. In contrast, TNF induced the expression of proinflammatory genes TNFSF10 and IL17 7-fold and 4-fold higher in patient cells than in healthy control cells. Increased TNF induced gene expression was accompanied by increased p65 nuclear translocation indicative of increased canonical IKK activation. These results suggest that aberrant expression of an alternately spliced NEMO isoform constitutes a novel autoinflammatory disease characterized by unbalanced NF-kB and IRF3 mediated responses to viral infection.
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Ruland, Jürgen, and Tak Mak. "MALT1 Is an Essential Regulator of Antigen Receptor Mediated NF-kappaB Activation." Blood 104, no. 11 (November 16, 2004): 347. http://dx.doi.org/10.1182/blood.v104.11.347.347.
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Abstract The translocation t(11;18)(q21;q21) is the most common chromosomal abnormality in lymphomas of mucosa associated lymphoid tissue (MALT lymphoma). Molecular cloning of the breakpoint demonstrated that t(11;18) leads to the fusion of the IAP2 gene to the novel gene MALT1. MALT1 encodes a paracaspase that can bind to BCL10 but its cellular function was unknown. We have previously demonstrated that BCL10, which is involved in independent MALT lymphoma translocations, functions as a positive regulator of lymphocyte proliferation. BCL10 connects antigen receptor signaling in B and T cells to NF-kappa B activation. To identify the physiological role of MALT1 we have now disrupted the MALT1 gene in mice. Here we are using both MALT1 and BCL10 deficient animals and show that MALT1 is essential for functional T cell activation, proliferation and cytokine production in response to T cell receptor (TCR) ligation. Similar to BCL10 we found MALT1 to be strictly required for signal-specific NF-kappaB activation in response to TCR ligation but not for TNF-alpha or IL-1 signaling. MALT1 operates downstream of BCL10, controls the catalytic activity of IKK and additionally regulates signaling of p38 and JNK MAP kinases. Moreover we found MALT1 to be critical for B1 and marginal zone (MZ) B cell development. In contrast to BCL10 disruption however, inactivation of MALT1 has only mild effects on conventional B2 B cell activation, BCR induced NF-kappaB signaling in splenic B lymphocytes and does not cause defects during embryonic development. Thus, MALT1 is an essential regulator of BCL10 mediated NF-kB induction that is differentially required depending on the cellular context. Together these data support the view that normal expression of BCL10 and MALT1 are necessary for controlled NF-kB signaling in lymphocytes whereas deregulation of either BCL10 or MALT1 through chromosomal translocation results in increased lymphocyte growth and survival
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Biswas, Polash Kumar, Sang Rok Park, Jongyub An, Kyung Min Lim, Ahmed Abdal Dayem, Kwonwoo Song, Hye Yeon Choi, et al. "The Orphan GPR50 Receptor Regulates the Aggressiveness of Breast Cancer Stem-like Cells via Targeting the NF-kB Signaling Pathway." International Journal of Molecular Sciences 24, no. 3 (February 1, 2023): 2804. http://dx.doi.org/10.3390/ijms24032804.
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The expression of GPR50 in CSLC and several breast cancer cell lines was assessed by RT-PCR and online platform (UALCAN, GEPIA, and R2 gene analysis). The role of GPR50 in driving CSLC, sphere formation, cell proliferation, and migration was performed using shGPR50 gene knockdown, and the role of GPR50-regulated signaling pathways was examined by Western blotting and Luciferase Assay. Herein, we confirmed that the expression of G protein-coupled receptor 50 (GPR50) in cancer stem-like cells (CSLC) is higher than that in other cancer cells. We examined that the knockdown of GPR50 in CSLC led to decreased cancer properties, such as sphere formation, cell proliferation, migration, and stemness. GPR50 silencing downregulates NF-kB signaling, which is involved in sphere formation and aggressiveness of CSLC. In addition, we demonstrated that GPR50 also regulates ADAM-17 activity by activating NOTCH signaling pathways through the AKT/SP1 axis in CSLC. Overall, we demonstrated a novel GPR50-mediated regulation of the NF-κB-Notch signaling pathway, which can provide insights into CSLC progression and prognosis, and NF-κB-NOTCH-based CSLC treatment strategies.
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Murphy, Michael, Xiong Yanbao, and Andrei Medvedev. "Pellino-1 and Pellino-3b in endotoxin tolerance and TLR signaling (INM9P.458)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 189.11. http://dx.doi.org/10.4049/jimmunol.192.supp.189.11.
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Abstract Endotoxin tolerance (ET) reprograms Toll-like receptor (TLR) 4-inducible responses in monocytes and macrophages by attenuating expression of pro-inflammatory cytokines without suppressing anti-inflammatory cytokines and antimicrobial effectors. We previously demonstrated deficient LPS-mediated TLR4-driven activation of IL-1 receptor-associated kinase (IRAK) 4 and IRAK1 as critical hallmarks of endotoxin tolerance. E3 ubiquitin ligases Pellino regulate TLR signaling pathways via modifications of IRAK kinases. We examined the impact of ET on Pellino-1/3b expression and utilized overexpression and gene ablation approaches to establish their functions in TLR2/4 signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in control THP-1 cells and human monocytes. Endotoxin-tolerant cells showed attenuated expression of Pellino-1 but maintained increased levels of Pellino-3b proteins. Overexpression of Pellino-1 in 293/TLR4/MD2 and 293/TLR2 cells enhanced Pam3Cys- and LPS-mediated NF-κB activation and cytokine gene expression, while Pellino-1 ablation reduced these responses demonstrating Pellino-1 as a positive regulator of TLR4 signaling. Pellino-3b overexpression reduced, whereas Pellino-3b gene knockdown increased TLR2/4-driven NF-kB activation and expression of inflammatory cytokines, establishing it as a negative regulator. These data suggest non-redundant functions for Pellino-1 and Pellino-3b within the TLR4 signaling pathways and implicate their role in ET.
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Gautam, SC, KR Pindolia, CJ Noth, N. Janakiraman, YX Xu, and RA Chapman. "Chemokine gene expression in bone marrow stromal cells: downregulation with sodium salicylate." Blood 86, no. 7 (October 1, 1995): 2541–50. http://dx.doi.org/10.1182/blood.v86.7.2541.2541.
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Abstract Chemotactic cytokines, chemokines, have been shown to influence the proliferation of hematopoietic progenitor cells. Thus, regulation of chemokine production by bone marrow accessory cells is a critical aspect of stromal cell regulation of hematopoiesis. We have previously reported that monocyte chemotactic protein-1 (MCP-1 or MCP-1/JE) and interferon inducible protein 10 kD (IP-10) are both induced in murine bone marrow stromal cells +/(+)-1.LDA11 after stimulation with the inflammatory agents interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), or lipopolysaccharide (LPS). In the present study, we have investigated the effect of sodium salicylate, an antiinflammatory agent, on the IL-1 alpha-induced expression of MCP-1/JE and IP-10 genes in stromal cells. Sodium salicylate attenuates the levels of MCP-1/JE and IP-10 mRNA in a concentration- and time-dependent manner. The suppression of MCP-1/JE mRNA is reversible, whereas IP-10 mRNA expression is more or less irreversibly affected as its recovery from the effect of sodium salicylate is slow and partial. Sodium salicylate-mediated suppression of mRNA expression is attributable neither to de novo synthesis of intermediary protein(s) nor to the destabilization of mature mRNA transcripts. On the other hand, sodium salicylate downregulates the transcriptional activity of both genes. Furthermore, IL-1 alpha induces activation of transcription factor nuclear factor (NF)-kB, and sodium salicylate suppresses it in a dose-dependent manner. We conclude that while posttranscriptional events remain unaffected, inhibition of NF-kB activation by sodium salicylate may account for the suppression of chemokine gene expression at the transcriptional level.
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Gautam, SC, KR Pindolia, CJ Noth, N. Janakiraman, YX Xu, and RA Chapman. "Chemokine gene expression in bone marrow stromal cells: downregulation with sodium salicylate." Blood 86, no. 7 (October 1, 1995): 2541–50. http://dx.doi.org/10.1182/blood.v86.7.2541.bloodjournal8672541.
Full textAbstract:
Chemotactic cytokines, chemokines, have been shown to influence the proliferation of hematopoietic progenitor cells. Thus, regulation of chemokine production by bone marrow accessory cells is a critical aspect of stromal cell regulation of hematopoiesis. We have previously reported that monocyte chemotactic protein-1 (MCP-1 or MCP-1/JE) and interferon inducible protein 10 kD (IP-10) are both induced in murine bone marrow stromal cells +/(+)-1.LDA11 after stimulation with the inflammatory agents interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), or lipopolysaccharide (LPS). In the present study, we have investigated the effect of sodium salicylate, an antiinflammatory agent, on the IL-1 alpha-induced expression of MCP-1/JE and IP-10 genes in stromal cells. Sodium salicylate attenuates the levels of MCP-1/JE and IP-10 mRNA in a concentration- and time-dependent manner. The suppression of MCP-1/JE mRNA is reversible, whereas IP-10 mRNA expression is more or less irreversibly affected as its recovery from the effect of sodium salicylate is slow and partial. Sodium salicylate-mediated suppression of mRNA expression is attributable neither to de novo synthesis of intermediary protein(s) nor to the destabilization of mature mRNA transcripts. On the other hand, sodium salicylate downregulates the transcriptional activity of both genes. Furthermore, IL-1 alpha induces activation of transcription factor nuclear factor (NF)-kB, and sodium salicylate suppresses it in a dose-dependent manner. We conclude that while posttranscriptional events remain unaffected, inhibition of NF-kB activation by sodium salicylate may account for the suppression of chemokine gene expression at the transcriptional level.
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Fang, Jing, Brenden Barker, Lyndsey Bolanos, Xiaona Liu, Andres Jerez, Hideki Makishima, Dinesh S. Rao, et al. "SQSTM1/p62 Is a Necessary Cofactor In MDS/AML With Deletion Of Mir-146a." Blood 122, no. 21 (November 15, 2013): 747. http://dx.doi.org/10.1182/blood.v122.21.747.747.
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Abstract Deletions involving chromosome 5 (del(5q)) are the most common genetic abnormalities in Myelodysplastic Syndrome (MDS) and secondary Acute Myeloid Leukemia (AML). Chromosome 5q deletions extending beyond q34 portend a worse overall survival, are associated with high-risk (HR) disease, and exhibit significant downregulation of miR-146a, a gene residing on the extended deleted region on 5q34. Additional evidence linking miR-146a loss to HR del(5q) MDS/AML comes from mouse genetic studies; miR-146a-/- mice develop a myeloid proliferative disease and myeloid tumors, in part by derepression of TNFR associated factor 6 (TRAF6) and persistent NF-kB activation. To determine the contribution of miR-146a deficiency to HR MDS/AML, we first examined hematopoietic stem/progenitor cells (HSPC) from miR-146a-/- mice. miR-146a-/- HSPC are highly proliferative, and exhibit increased cell survival and altered HSC fitness. In addition, genetic and/or pharmacologic inhibition of TRAF6/NF-kB signaling impairs cell cycle progression and preferentially leads to apoptosis of malignant miR-146a-/- HSPC. Although inhibiting the TRAF6/NF-kB axis may represent a therapeutic opportunity in miR-146alow MDS/AML patients, unfortunately, NF-kB inhibitors in clinical trials have been disappointing and ones for TRAF6 do not exist. Chromosome deletions that target tumor suppressor genes also involve multiple neighboring genes, such as with del(5q), and loss of certain neighboring genes may expose cancer-specific vulnerabilities. To overcome the limitations of NF-kB inhibitors and identify novel therapeutic targets, we examined the expression of all genes residing within the long arm on chr 5 (5q11-q35) from del(5q) MDS and control CD34+ cells and built molecular networks using GeneConnector functionality in NetWalker. A single major intrachromosomal NF-kB signaling node formed corresponding to the overexpressed chr 5q genes. Among the compensated/overexpressed genes residing on chr 5q and within the NF-kB node, SQSTM1/p62 (5q35) emerged as an obvious candidate as it is an essential cofactor for NF-kB activation by binding TRAF6. First, we evaluated the contribution of p62 to the malignant miR-146alow HSPC phenotype. Overexpression of p62 enhanced proliferation of miR-146a-/- HSPC by promoting G2/M cell cycle progression. Conversely, knockdown of p62 in miR-146a-/- HSPC led to cell cycle arrest and rescued defective myeloid engraftment in competitive HSC transplantation assays, suggesting p62 is required in miR-146alow leukemic cells. Furthermore, the importance of p62 was confirmed in MDS/AML cell lines and patient samples. RNAi-mediated knockdown of p62 resulted in a G2/M cell cycle arrest, reduced cell survival, and impaired leukemic progenitor function, underscoring the importance of p62 in MDS/AML. In addition, interfering with p62-TRAF6 binding by overexpressing a small peptide corresponding to the p62-TRAF6 binding interface suppressed TRAF6-mediated NF-kB activation, and similarly inhibited cell cycle progression and induced apoptosis of human miR-146alow leukemic cells. Collectively, these findings reveal an intrachromosomal gene network that not only drives HR del(5q) myeloid malignancies, but also exposes them to cancer-specific therapeutic vulnerability by disrupting the binding between p62 and TRAF6. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.
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Fang, Jing, Andres Jerez, Christopher Rasch, Lyndsey Bolanos, Jaroslaw P. Maciejewski, and Daniel T. Starczynowski. "Haploinsufficiency of Mir-146a in High-Risk Del(5q) MDS/AML Requires an Intrachromosomal Gene Network Involving p62/TRAF6/NF-κB." Blood 120, no. 21 (November 16, 2012): 557. http://dx.doi.org/10.1182/blood.v120.21.557.557.
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Abstract Abstract 557 Deletion of chromosome 5q (del(5q)) is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Commonly deleted regions (CDR) have been mapped to 5q31.1 and 5q33.1 in del(5q) AML and MDS, respectively. Although there has been extensive efforts to identify candidate genes within the CDRs and decipher which genes contribute to the high-risk versus low-risk phenotypes, more recent findings indicate that deletions involving the telomeric bands of 5q are associated with more aggressive forms of del(5q) MDS/AML (Jerez et al., JCO, 2012). To determine which genes on the telomeric extremes of chr 5q contribute to the aggressive phenotype associated with these patients, we performed a gene expression and functional analysis of candidate tumor suppressor (TS) genes located between ∼5q34 to 5q35.3. Among the 7 candidate TS genes localized on the telomeric extreme, expression of miR-146a is significantly reduced in del(5q) MDS/AML marrow cells with extended deletions as compared to ones with shorter deletions. In addition, low expression of miR-146a correlates with increased marrow blasts and shorter overall survival of patients with del(5q). To investigate the effects of miR-146a on hematopoietic stem/progenitor cell (HSPC) function, we stably knocked down miR-146a in human CD34+ cord blood and mouse HSPC-enriched cells. HSPC cells with reduced miR-146a exhibited features of malignant transformation, including increased survival, proliferation, and colony replating in methylcellulose. The mechanism underlying reduced miR-146a expression was investigated by performing gene expression profiling on marrow cells from MDS/AML patients with extended and short del(5q). Gene Ontology and Gene Set Enrichment Analysis revealed that patients with low expression of miR-146a exhibited a significant increase in cell cycle, immune response, and NF-kB target genes. These findings are consistent with the model that reduced levels of miR-146a results in derepression of TRAF6, a signaling protein within the innate immune pathway and activator of NF-kB (Starczynowski et al., Nature Medicine 2010). TRAF6 forms a signaling complex with Sequestosome 1 (SQSTM1/p62), a gene located within the telomeric deleted region in del(5q) on band 5q34. Surprisingly, expression of p62 was not reduced, but rather overexpressed in del(5q) MDS/AML patients with the extended deletions and inversely correlated with miR-146a expression. The compensatory expression of p62 is due in part by loss of miR-146a. RNAi-mediated knockdown revealed that p62 and TRAF6 are essential for NF-kB activation and survival of human MDS/AML cell lines with low miR-146a expression. In summary, we identified that miR-146a is a TS-like gene in MDS/AML patients with extended deletions of chr 5q, and that reduced miR-146a expression increases HSPC survival/proliferation and NF-kB signaling via p62 and TRAF6. Therefore, we propose that inhibiting the p62/TRAF6/NFκB intrachromosomal gene network represents a novel targeting strategy in high-risk MDS/AML patients with extended chr 5q deletions. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
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Pessara, U., and N. Koch. "Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element." Molecular and Cellular Biology 10, no. 8 (August 1990): 4146–54. http://dx.doi.org/10.1128/mcb.10.8.4146-4154.1990.
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Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.
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Pessara, U., and N. Koch. "Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element." Molecular and Cellular Biology 10, no. 8 (August 1990): 4146–54. http://dx.doi.org/10.1128/mcb.10.8.4146.
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Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.
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Al-Rashed, Fatema, Reeby Thomas, Areej Al-Roub, Fahd Al-Mulla, and Rasheed Ahmad. "LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1." Molecules 25, no. 20 (October 14, 2020): 4709. http://dx.doi.org/10.3390/molecules25204709.
Full textAbstract:
Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.
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Alrashed, Fatema, Reeby Thomas, Areej Al-Roub, Fahd AL-Mulla, and Rasheed Ahmad. "LPS Induces GM-CSF Production by Breast Cancer MDA-MB-231 Cells via Long-Chain Acyl-CoA Synthetase 1." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 56.20. http://dx.doi.org/10.4049/jimmunol.206.supp.56.20.
Full textAbstract:
Abstract Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a monomeric glycoprotein that has been implicated in the tumor growth and progression of different types of cancer. GM-CSF is produced by various non-immune cells including MDA-MB-231 in response to various stimuli. However, the role of lipopolysaccharide (LPS) in the regulation of GM-CSF in MDA-MB-231 breast cancer cells so far remains unclear. Herein, we asked whether LPS could induce GM-CSF production in MDA-MB-231 cells, and if so, which signaling pathway was involved. MDA-MB-231 cells were treated with LPS or tumor necrosis factor alpha (TNF-α; positive control), and GM-CSF expression levels were determined by qRT-PCR, ELISA, and confocal microscopy. Phosphorylation of the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-kB) signaling proteins were evaluated by flow cytometry. Our results show that LPS induces GM-CSF expression at both mRNA and protein levels in MDA-MBA-231 cells. Inhibition of acyl-CoA synthetase 1 (ACSL1) activity in the cells with triacsin C significantly reduces the secretion of GM-CSF. Furthermore, the inhibition of ACSL1 activity significantly blocks the LPS-mediated phosphorylation of p38 MAPK, MEK1/2, extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-kB) in the cells. These findings provide the first evidence that LPS induces ACSL1-dependent GM-CSF gene expression in MDA-MB-231 breast cancer cells, which requires the activation of p38 MAPK, MEK1/2, ERK1/2, JNK, and NF-kB.
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Otsuyama, Ken-ichiro, Jakia Amin, Emi Uchida, Saeid Abroun, Karim Shamsasenjan, Khademul Islam, Hideki Asaoku, and Michio M. Kawano. "Baicalein Combined with Dexamethasone Can Induce the Marked PPARß-Mediated Suppression of Growth and Survival in Human Myeloma Cells." Blood 108, no. 11 (November 16, 2006): 5085. http://dx.doi.org/10.1182/blood.v108.11.5085.5085.
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Abstract PPAR (Peroxisome proliferator-activated receptor) ß is ubiquitously expressed in all cells and considered to be involved in the lipid metabolism and regulating the inflammatory response and cell proliferation. However, it remain to be clarified the role of PPARß in human myeloma cells. In order to identify the possible ligands for PPARß, we constructed the PPARß response element (PPREß)-lusiferase reporter gene and performed the reporter assay in Hela cell. We found that adrenal cortex hormones (DHEA and DHEA-S etc), dexamethasone (Dex) and baicalein augmented the expression of reporter gene. As already reported by us, baicalein, one of the major flavonoids in Scutellaria baicalensis (Chinese Skullcap), has the suppressive effects on cell proliferation and angiogenesis. Furthermore, baicalein as well as Dex upregulated the expression of PPARß target genes such as ILK and PPARg in myeloma cell lines. Moreover the combination of baicalein with Dex showed cooperative effect on the growth suppression in primary myeloma cells as well as myeloma cell lines. The expression of NF-kB target genes such as IL-6 was also markedly suppressed with the treatment with baiacalein and Dex in U266 cells. Since U266 cells constitutively express RelA(p65), RelB and p50 in the nucleus, we examined whether ligand (baicalein or Dex)-stimulated PPAR ß interacted with RelA in the nucleus of U266 cells or not. We detected physical interaction of PPARß with RelA in the nuclear fraction of U266 cells after stimulation with baicalein or Dex, and also confirmed that baicalein and Dex suppressed the DNA binding of RelA to kB site in the p65 ELISA. Therefore, these data suggest that baicalein combined with Dex can stimulate PPARß-mediated growth suppression in myeloma cells possibly through nuclear interaction between PPARß and RelA (NF-kB) more effectively.
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Cheng, Qilai, Meixiang Liao, Haibo Hu, Hongliang Li, and Longhuo Wu. "Asiatic Acid (AA) Sensitizes Multidrug-Resistant Human Lung Adenocarcinoma A549/DDP Cells to Cisplatin (DDP) via Downregulation of P-Glycoprotein (MDR1) and Its Targets." Cellular Physiology and Biochemistry 47, no. 1 (2018): 279–92. http://dx.doi.org/10.1159/000489806.
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Background/Aims: P-glycoprotein (P-gp, i.e., MDR1) is associated with the phenotype of multidrug resistance (MDR) and causes chemotherapy failure in the management of cancers. Searching for effective MDR modulators and combining them with anticancer drugs is a promising strategy against MDR. Asiatic acid (AA), a natural triterpene isolated from the plant Centella asiatica, may have an antitumor activity. The present study assessed the reversing effect of AA on MDR and possible molecular mechanisms of AA action in MDR1-overexpressing cisplatin (DDP)-resistant lung cancer cells, A549/DDP. Methods: Human lung adenocarcinoma A549/DDP cells were either exposed to different concentrations of AA or treated with DDP, and their viability was measured by the MTT assay. A Rhodamine 123 efflux assay, immunofluorescent staining, ATPase assay, reverse-transcription PCR (RT-PCR), and western blot analysis were conducted to elucidate the mechanisms of action of AA on MDR. Results: Our results showed that AA significantly enhanced the cytotoxicity of DDP toward A549/DDP cells but not its parental A549 cells. Furthermore, AA strongly inhibited P-gp expression by blocking MDR1 gene transcription and increased the intracellular accumulation of the P-gp substrate Rhodamine 123 in A549/DDP cells. Nuclear factor (NF)-kB (p65) activity, IkB degradation, and NF-kB/p65 nuclear translocation were markedly inhibited by pretreatment with AA. Additionally, AA inhibited the MAPK–ERK pathway, as indicated by decreased phosphorylation of ERK1 and -2, AKT, p38, and JNK, thus resulting in reduced activity of the Y-box binding protein 1 (YB1) via blockage of its nuclear translocation. Conclusions: AA reversed P-gp-mediated MDR by inhibition of P-gp expression. This effect was likely related to downregulation of YB1, and this effect was mediated by the NF-kB and MAPK–ERK pathways. AA may be useful as an MDR reversal agent for combination therapy in clinical trials.
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Alrashed, Fatema, Zunair Ahmad, Reeby Thomas, Abdulwa Alghaith, Motasem Melhem, and Rasheed Ahmad. "Neutral sphingomyelinase 2 regulates inflammatory responses in monocytes/macrophages induced by TNF-α." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 59.26. http://dx.doi.org/10.4049/jimmunol.204.supp.59.26.
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Abstract Obesity is associated with elevated levels of TNF-α and proinflammatory CD11c monocytes/macrophages. TNF-α mediated dysregulation in the plasticity of monocytes/macrophages is concomitant with pathogenesis of several inflammatory diseases, including metabolic syndrome, but the underlying mechanisms are incompletely understood. Since neutral sphingomyelinase 2 (nSMase2; product of the sphingomyelin phosphodiesterase 3 gene, SMPD3) is a key enzyme for ceramide production involved in inflammation, we investigated whether the nSMase2 contributed to the inflammatory changes in the monocytes/macrophages induced by TNF-α. In this study, we demonstrate that the disruption of nSMase2 activity in monocytes/macrophages either by chemical inhibitor GW4869 or small interfering RNA (siRNA) against SMPD3 results in defects in the TNF-α mediated expression of CD11c. Furthermore, blockage of nSMase in monocytes/macrophages inhibited the secretion of IL-1b and MCP-1. However, inhibition aSMase activity did not attenuate CD11c expression and secretion of IL-1b and MCP-1. Phosphorylation of JNK, p38 and NF-κB resulting from TNF-α stimulation was also attenuated by the inhibition of nSMase. Moreover, NF-kB/AP-1 activity was blocked by the inhibition of nSMase2. Our human data show that SMPD3 gene expression was not only found to be elevated in obese individuals but also positively corelate with TNF-α elevated expression. These findings indicate that nSMase acts, in a part, as a master switch in the TNF-α mediated inflammatory responses in monocytes/macrophages.
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Varney, Melinda, Andres Jerez, Jing Fang, David Miller, Lyndsey Bolanos, Aly Karsan, Jaroslaw P. Maciejewski, and Daniel T. Starczynowski. "Tifab, a Novel Candidate Gene in Deletion Chromosome 5q, Contributes to Deregulation of NF-κB Signaling in MDS/AML." Blood 120, no. 21 (November 16, 2012): 2812. http://dx.doi.org/10.1182/blood.v120.21.2812.2812.
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Abstract Abstract 2812 Myelodysplastic syndromes (MDS) are hematologic disorders defined by blood cytopenias due to ineffective hematopoiesis, altered cytogenetics, and predisposition to acute myeloid leukemia (AML). The most common cytogenetic alteration in de novo and treatment-related MDS is deletion of chromosome 5q (del(5q)). There are two commonly deleted regions (CDR) mapped to chr 5q, however the gene(s) in these regions responsible for the manifestation of del(5q) MDS are not clearly defined. A search of annotated genes revealed that TRAF-interacting protein with forkhead-associated domain B (TIFAB), a known inhibitor of TRAF6 and a novel gene identified by an in silico search for TIFA-related genes, resides within the proximal CDR on band 5q31.1. We first determined whether TIFAB is expressed in normal hematopoietic stem/progenitor cell (HSPC) by qRT-PCR. We find that expression of TIFAB is enriched in human CD34+/CD38+ and mouse lineage-/cKit+ progenitors as compared to more differentiated populations, suggesting that it plays a role in normal HSPC function. To determine whether TIFAB is implicated in del(5q) MDS, we measured TIFAB expression in del(5q) MDS patients. According to a microarray analysis, TIFAB mRNA was significantly lower in CD34+cells isolated from MDS patients with del(5q) as compared with cells from MDS patients diploid at chr 5q (Pellagatti, et al., 2006). In an independent subset of patients, we confirmed that TIFAB expression was lower in marrow cells isolated from del(5q) MDS patients. Therefore, we hypothesize that TIFAB loss results in hematopoietic defects contributing to del(5q) MDS. To determine whether deletion of TIFAB affects hematopoiesis, we used lentiviral shRNAs to knockdown TIFAB mRNA in human cord blood CD34+ cells. To mimic haploinsufficiency of TIFAB in del(5q) MDS, we selected shRNAs that result in ∼50% knockdown of TIFAB mRNA and protein. Knockdown of TIFAB in human CD34+ cells results in increased survival, a competitive growth advantage, and altered hematopoietic progenitor function. Conversely, overexpression of TIFAB in human leukemia cell lines (THP1 and HL60) results in increased basal apoptosis, delayed G1/S-phase cell cycle progression, and impaired leukemic progenitor function in methylcellulose. Since TIFAB is predicted to regulate TRAF6, we examined the role of TIFAB on TRAF6 signaling. TIFAB suppressed TRAF6 lysine (K)-63 autoubiquitination (a measure of TRAF6 activity), and decreased total TRAF6 protein levels, suggesting that TIFAB may simultaneously inhibit TRAF6 function and protein expression. Consistent with this finding, TIFAB suppressed lipopolysaccharide-induced (TRAF6-dependent) NF-kB activation, but not TNF-induced (TRAF6-independent) NF-kB activation. TIFAB-mediated inhibition of TRAF6 also coincided with reduced phospho-IKK-beta (a measure of NF-kB activation) in leukemic cells. In summary, we have identified TIFAB as a novel del(5q) MDS/AML gene involved in regulating HSPC survival, progenitor function, and cell cycle. We propose that haploinsufficiency of TIFAB results in malignant clonal cell expansion and may contribute to the MDS/AML phenotype as a consequence of increased TRAF6-mediated activation of NF-kB. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.
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Ellegast, Jana M., Gabriela Alexe, Amanda Hamze, Shan Lin, Maxim Pimkin, Linda Ross, Neekesh V. Dharia, et al. "Unleashing Cell-Intrinsic Inflammation As a Strategy to Kill AML Blasts." Blood 138, Supplement 1 (November 5, 2021): 3305. http://dx.doi.org/10.1182/blood-2021-151511.
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Abstract Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To test the hypothesis, we analyzed three large, independent data collections from primary acute myeloid leukemia (AML) samples and identified an AML subgroup of approximately 40% of all samples enriched for immune and inflammatory pathways. Moreover, we observed a positive correlation between the enrichment of inflammatory response and a monocytic lineage signature in these primary AML samples. To discover genetic vulnerabilities in AML cells implicated in inflammatory pathways, we integrated data from the Cancer Dependency Map on 789 cancer cell lines with two independent genome-wide screens. We identified the immune modulator interferon regulatory factor 2 binding protein 2 (IRF2BP2) as a selective dependency in AML. We validated AML cell dependency on IRF2BP2 with orthogonal genetic and degradation approaches in vitro and in vivo. Genetic perturbation and degradation of IRF2BP2 translated into a reduction of AML cell line viability and decreased colony formation capacity in vitro and impaired leukemia progression in vivo in AML cell line xenografts. Moreover, inducible knock-out of IRF2BP2 significantly attenuated disease burden in multiple patient-derived xenograft models of AML and prolonged survival. Mechanistically, IRF2BP2 repression induced cell death with hallmarks of apoptosis, evidenced by an increase in annexin V/PI-positive cells and induction of cleaved caspase 3 and 8. To decipher the role of IRF2BP2 in inflammatory signaling, we studied its localization on chromatin and its relationship to histone marks and RNA polymerase II binding in AML cells. We determined that IRF2BP2 binds at both enhancers and promotors. Global gene expression profiling, performed six hours after degradation of IRF2BP2, identified significantly more genes increased than decreased in expression. Most upregulated genes were bound by IRF2BP2 at baseline and gained H3K27ac at enhancers with IRF2BP2 degradation. All told, our data support a role for IRF2BP2 as a transcriptional repressor in AML. Gene set enrichment analysis of the genes directly regulated by IRF2BP2 identified immune response signatures as the top enriched gene sets, with genes regulated by NF-kB in response to TNFa being the most significantly enriched. We thus hypothesized that IRF2BP2 is a repressor of NF-kB mediated TNFa signaling that, when acutely perturbed, leads to leukemia cell death. Indeed, we observed an activation of NF-κB signaling in a luciferase reporter assay, an increase in nuclear NF-kB (RELA) protein levels, and a gain in NF-κB chromatin binding following degradation of IRF2BP2 in AML cells. Moreover, a mutant "super-repressor" allele of IkBa rescued the impaired cell growth upon IRF2BP2 perturbation in AML cells, supporting cell death associated with IRF2BP2 loss being mediated through activation of NF-kB signaling. In addition, we identified IL-1ß as an enhancer of the inflammatory response repressed by IRF2BP2. In summary, we have demonstrated that IRF2BP2 represses IL-1ß/TNFα signaling via NF-κB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. Disclosures Dharia: Genentech: Current Employment. Benajiba: Gilead: Research Funding; Pfizer: Research Funding. Gray: Gatekeeper, Syros, Petra, C4, Allorion, Jengu, B2S, Inception and Soltego: Consultancy, Research Funding. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding.
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Kuncio, Gerald S., Mariya Tsyganskaya, Jianling Zhu, Shu-Ling Liu, Laszlo Nagy, Vilmos Thomazy, Peter J. A. Davies, and Mark A. Zern. "TNF-α modulates expression of the tissue transglutaminase gene in liver cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 274, no. 2 (February 1, 1998): G240—G245. http://dx.doi.org/10.1152/ajpgi.1998.274.2.g240.
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One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-α, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-α increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-α, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-α. Gel shift experiments using nuclear extracts from TNF-α-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-κB motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-κB sequence showed an attenuated response to TNF-α treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a trangenic mouse line containing the mouse tTG promoter driving the β-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-α. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-α when those cells overexpress a transdominant, negative mutant NF-κB subunit. Because TNF-α expression is upregulated in hepatic inflammation, the data suggest TNF-α-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.