NF-κB Subunit c-Rel Research Articles

Glut3 promotes cellular O-GlcNAcylation as a distinctive tumor-supportive feature in Treg cells.

Regulatory T cells (Tregs) establish dominant immune tolerance but obstruct tumor immune surveillance, warranting context-specific mechanistic insights into the functions of tumor-infiltrating Tregs (TIL-Tregs). We show that enhanced posttranslational O-linked N-acetylglucosamine modification(O-GlcNAcylation) of cellular factors is a molecular feature that promotes a tumor-specific gene expression signature and distinguishes TIL-Tregs from their systemic counterparts. We found that altered glucose utilization through the glucose transporter Glut3 is a major facilitator of this process. Treg-specific deletion of Glut3 abrogates tumor immune tolerance, while steady-state immune homeostasis remains largely unaffected in mice. Furthermore, by employing mouse tumor models and human clinical data, we identified the NF-κB subunit c-Rel as one such factor that, through Glut3-dependent O-GlcNAcylation, functionally orchestrates gene expression in Tregs at tumor sites. Together, these results not only identify immunometabolic alterations and molecular events contributing to fundamental aspects of Treg biology, specifically at tumor sites but also reveal tumor-specific cellular properties that can aid in the development of Treg-targeted cancer immunotherapies.

NF-κB subunits RelA and c-Rel selectively control CD4+ T cell function in multiple sclerosis and cancer.

The outcome of cancer and autoimmunity is often dictated by the effector functions of CD4+ conventional T cells (Tconv). Although activation of the NF-κB signaling pathway has long been implicated in Tconv biology, the cell-autonomous roles of the separate NF-κB transcription-factor subunits are unknown. Here, we dissected the contributions of the canonical NF-κB subunits RelA and c-Rel to Tconv function. RelA, rather than c-Rel, regulated Tconv activation and cytokine production at steady-state and was required for polarization toward the TH17 lineage in vitro. Accordingly, RelA-deficient mice were fully protected against neuroinflammation in a model of multiple sclerosis due to defective transition to a pathogenic TH17 gene-expression program. Conversely, Tconv-restricted ablation of c-Rel impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. Moreover, Tconv required c-Rel for the response to PD-1-blockade therapy. Our data reveal distinct roles for canonical NF-κB subunits in different disease contexts, paving the way for subunit-targeted immunotherapies.

The transcription factor NF-κB orchestrates nucleosome remodeling during the primary response to Toll-like receptor 4 signaling

Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regionswas conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.

SALMONELLA INFECTION: INTERPLAY BETWEEN THE T3SSS EFFECTORS AND NF-ΚB SIGNALING PATHWAY

Salmonella is an important foodborne pathogen that can evade host immune defense by evolving unique mechanisms. Salmonella manipulate host cell various signaling pathways by delivering specific effectors into target cells to establish infection. The nuclear factor-κB (NF-κB) is an important nuclear transcription factor that regulates the host immune system in Salmonella infection. The Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) encode type III secretion systems (T3SSs), effectors that are associated with the NF-κB signaling pathway through regulate host inflammation response. SPI-1 effectors SipA, SopE, SopE2, and SopB all can activate NF-κB signaling pathway to facilitate Salmonella invasion and intracellular carriage. Studies have shown that T3SS1 and/or T3SS2 effectors such as GtgA, GogA and PipA contain two histidine residues and have metalloprotease activity to control Salmonella replication. These zinc metalloproteases redundantly target the NF-κB subunits p65, RelB, and c-Rel, whereas GogA and GtgA only inhibit NF-κB-dependent gene transcription. The T3SS2 effectors SseK1, SseK2, and SseK3 are death domain-containing proteins with N-linked glycosyltransferase characteristics that can inhibit NF-κB activity by inhibiting IκBα phosphorylation in TNF- α-treated 293ET cells. Among them, SseK1 and SseK3 also suppress Salmonella-induced NF-κB activity in macrophages. SseK3-mediated inhibition of the NF-B signaling pathway is not required for protein 32 containing a tripartite E3-ubiquitin ligase motif. In addition, the SPI-2 T3SS effector SpvD inhibits NF-κB activity by preventing nuclear translocation of p65 through interaction with Exportin-2, but this does not affect IκBα degradation, which ultimately leads to systemic Salmonella growth. However, other effectors SptP, AvrA, IpaJ, SspH1, GtgA, GogA, and SPI-2 encoded SseL, SpvB, SseK1, and GogB all can effectively inhibit NF-κB signaling pathway, and contribute to Salmonella intracellular replication and virulence. In this mini-review, we summarize the special mechanism how NF-κB signaling pathway is regulated by Salmonella T3SSs effectors in the persistent infection of Salmonella, which will further elucidate the pathogenesis of Salmonella.

Atypical IκB Bcl3 enhances the generation of the NF-κB p52 homodimer

The NF-κB family of dimeric transcription factors regulate diverse biological functions. Their cellular expression profiles differ, which lead to different concentrations in different cell/tissue types. Although the activation mechanisms of different NF-κB dimers have been widely investigated, there is limited information on specific NF-κB dimers’ formation. The NF-κB p52:p52 homodimer regulates an important subset of target genes in cancer cells; however, the molecular mechanism of the generation of this specific homodimer remains unclear. Our study has revealed that the atypical IκB protein, Bcl3, plays an essential role in enhancing the p52:p52 homodimer population which is a unique mechanism to p52 within the NF-κB family. p52 was shown to heterodimerize with four other NF-κB subunits (RelA, RelB, cRel, and p50); all heterodimers, except p52:p50, are significantly more stable than the p52:p52 homodimer. Bcl3 is able to compete with all other NF-κB subunits in cells for efficient p52:p52 homodimer formation which consequently leads to the upregulation of target genes that are involved in cell proliferation, migration, and inflammation, which explain why aberrant activation of Bcl3 and p52 leads to cancer.

Dipeptidyl peptidase-8 induces sorafenib resistance via binding with c-Rel to mediate NF-κB signaling in hepatocellular carcinoma.

Sorafenib is the important first-standard drug for patients with advanced hepatocellular carcinoma (HCC). A major obstacle to successful treatment is sorafenib resistance. However, the mechanism of sorafenib resistance is unclear. The present study aimed to determine the involvement of dipeptidyl peptidase-8 (DPP8) in sorafenib resistance. DPP8 expression was detected using quantitative real-time PCR (qPCR) and western blot analysis. The effect of DPP8 on sorafenib resistance was examined using terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL), colony formation, flow cytometry, luciferase reporter, immunofluorescence, and immunoprecipitation (IP) assays. We found that DPP8 mRNA and protein levels were dramatically upregulated in HCC. Gene set enrichment analysis (GSEA) illustrated that DPP8 might be involved in apoptosis regulation. Downregulation of DPP8 substantially promoted the sensitivity of HCC cells to sorafenib. Further analysis showed that DPP8 might regulate nuclear factor kappa B (NF-κB) signaling, which was confirmed using a luciferase reporter assay. Downregulation of DPP8 decreased the expression levels of downstream genes of the NF-κB pathway. IP showed that DPP8 can interact with NF-κB subunit c-Rel, an important protein of NF-κB signaling. Finally, a drug combination of sorafenib and Val-boroPro induced higher mortality of HCC cells than sorafenib alone in DPP8-upregulated cells. Our findings indicated that using the inhibitor Val-boroPro might be a promising method to enhance sorafenib sensitivity in advanced HCC.

The NF-κB Transcription Factor c-Rel Modulates Group 2 Innate Lymphoid Cell Effector Functions and Drives Allergic Airway Inflammation.

Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and orchestration of early type 2 immune responses. Upon tissue damage, ILC2s are activated by alarmins such as IL-33 and rapidly secrete large amounts of type 2 signature cytokines. ILC2 activation is governed by a network of transcriptional regulators including nuclear factor (NF)-κB family transcription factors. While it is known that activating IL-33 receptor signaling results in downstream NF-κB activation, the underlying molecular mechanisms remain elusive. Here, we found that the NF-κB subunit c-Rel is required to mount effective innate pulmonary type 2 immune responses. IL-33-mediated activation of ILC2s in vitro as well as in vivo was found to induce c-Rel mRNA and protein expression. In addition, we demonstrate that IL-33-mediated activation of ILC2s leads to nuclear translocation of c-Rel in pulmonary ILC2s. Although c-Rel was found to be a critical mediator of innate pulmonary type 2 immune responses, ILC2-intrinsic deficiency of c-Rel did not have an impact on the developmental capacity of ILC2s nor affected homeostatic numbers of lung-resident ILC2s at steady state. Moreover, we demonstrate that ILC2-intrinsic deficiency of c-Rel alters the capacity of ILC2s to upregulate the expression of ICOSL and OX40L, key stimulatory receptors, and the expression of type 2 signature cytokines IL-5, IL-9, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Collectively, our data using Rel −/− mice suggest that c-Rel promotes acute ILC2-driven allergic airway inflammation and suggest that c-Rel may contribute to the pathophysiology of ILC2-mediated allergic airway disease. It thereby represents a promising target for the treatment of allergic asthma, and evaluating the effect of established c-Rel inhibitors in this context would be of great clinical interest.

Intracellular AGR2 transduces PGE2 stimuli to promote epithelial–mesenchymal transition and metastasis of colorectal cancer

Human anterior gradient homolog 2 (AGR2) reportedly acts as an oncogene in multiple types of cancers. As a secreted protein, the oncogenic roles of extracellular AGR2 have been the focus of the increasing number of studies. In contrast, the oncological functions of intracellular AGR2 (iAGR2) remain elusive. Here, we report that intracellular AGR2 (iAGR2) is sufficient to promote CRC metastasis. iAGR2 binds to KDEL receptors (KDELRs) via its KTEL motif to activate downstream Gs-PKA signaling. Activated PKA upregulates the expression of NF-κB subunit c-Rel (REL) and acetylates histone H3 at lysine 9 (H3K9ac) to promote the transcription of SNAIL and SLUG. AGR2 can be upregulated by prostaglandin E2 (PGE2) via EP4-PI3K-AKT pathway and is indispensable for PGE2-induced CRC metastasis. AGR2 knockdown enhances therapeutic effects of a COX-2 inhibitor, celecoxib, in CRC metastasis. Collectively, our study reveals a promoting role and molecular mechanisms of iAGR2 in CRC metastasis and uncovers a new tumor microenvironment signal regulating AGR2 expression, which may provide new targets for treating metastatic CRC.

NF-κB inhibition in keratinocytes causes RIPK1-mediated necroptosis and skin inflammation.

Tumor necrosis factor receptor 1 (TNFR1) activates NF-κB-dependent pro-inflammatory gene expression, but also induces cell death by triggering apoptosis and necroptosis. Inhibition of inhibitor of NF-κB kinase (IKK)/NF-κB signaling in keratinocytes paradoxically unleashed spontaneous TNFR1-mediated skin inflammation in mice, but the underlying mechanisms remain poorly understood. Here, we show that TNFR1 causes skin inflammation in mice with epidermis-specific knockout of IKK2 by inducing receptor interacting protein kinase 1 (RIPK1)-dependent necroptosis, and to a lesser extent also apoptosis, of keratinocytes. Combined epidermis-specific ablation of the NF-κB subunits RelA and c-Rel also caused skin inflammation by inducing TNFR1-mediated keratinocyte necroptosis. Contrary to the currently established model that inhibition of NF-κB-dependent gene transcription causes RIPK1-independent cell death, keratinocyte necroptosis, and skin inflammation in mice with epidermis-specific RelA and c-Rel deficiency also depended on RIPK1 kinase activity. These results advance our understanding of the mechanisms regulating TNFR1-induced cell death and identify RIPK1-mediated necroptosis as a potent driver of skin inflammation.

C-Rel orchestrates energy-dependent epithelial and macrophage reprogramming in fibrosis.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-β1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.

Inhibition of IL-2 or NF-κB Subunit c-Rel-Dependent Signaling Inhibits Expansion of Regulatory T Cells During Acute Friend Retrovirus Infection.

In retroviral infections, different immunological mechanisms are involved in the development of a chronic infection. In the Friend virus (FV) model, regulatory T cells (Tregs) were found to induce CD8+ T cell dysfunction before viral clearance is achieved and thus contribute to viral chronicity. Although studied for decades, the exact suppressive mechanisms of Tregs in the FV model remain elusive and an unavailable therapeutic target. However, extracellular IL-2 and intracellular NF-κB signaling were shown to be important pathways for Treg expansion and activation. Therefore, we decided to focus on these two pathways to test therapeutic approaches inhibiting Treg activation during FV infection. In this study, we show that the inhibition of either IL-2 or the NF-κB subunit c-Rel, impaired Treg expansion and activation at 2 weeks post-FV infection. Total numbers of Tregs as well as activated Tregs were reduced in FV-infected mice after treatment with anti-IL-2 antibodies or the c-Rel blocking reagent pentoxifylline. Surprisingly, this did not affect the expansion or function of virus-specific CD8+ T cells nor viral loads in the spleen. However, our data suggest that neutralization of IL-2 as well as blocking c-Rel efficiently inhibits virus-induced Treg expansion.

Regulation of B-cell function by NF-kappaB c-Rel in health and disease.

B cells mediate humoral immune response and contribute to the regulation of cellular immune response. Members of the Nuclear Factor kappaB (NF-κB) family of transcription factors play a major role in regulating B-cell functions. NF-κB subunit c-Rel is predominantly expressed in lymphocytes, and in B cells, it is required for survival, proliferation, and antibody production. Dysregulation of c-Rel expression and activation alters B-cell homeostasis and is associated with B-cell lymphomas and autoimmune pathologies. Based on its essential roles, c-Rel may serve as a potential prognostic and therapeutic target. This review summarizes the current understanding of the multifaceted role of c-Rel in B cells and B-cell diseases.

NF-κB c-Rel Dictates the Inflammatory Threshold by Acting as a Transcriptional Repressor.

SummaryNF-κB/Rel family of transcription factors plays a central role in initiation and resolution of inflammatory responses. Here, we identified a function of the NF-κB subunit c-Rel as a transcriptional repressor of inflammatory genes. Genetic deletion of c-Rel substantially potentiates the expression of several TNF-α-induced RelA-dependent mediators of inflammation. v-Rel, the viral homologue of c-Rel, but not RelB, also possesses this repressive function. Mechanistically, we found that c-Rel selectively binds to the co-repressor HDAC1 and competitively binds to the DNA mediating HDAC1 recruitment to the promoters of inflammatory genes. A specific point mutation at tyrosine25 in c-Rel's DNA-binding domain, for which a missense single nucleotide variation (Y25H) exists in humans, completely abrogated its ability to bind DNA and repress TNF-α-induced, RelA-mediated transcription. Our findings reveal that the transactivator NF-κB subunit c-Rel also plays a role as a transcriptional repressor in the maintenance of inflammatory homeostasis.

Evidence for Anti-Inflammatory Activity of Isoliquiritigenin, 18β Glycyrrhetinic Acid, Ursolic Acid, and the Traditional Chinese Medicine Plants Glycyrrhiza glabra and Eriobotrya japonica, at the Molecular Level.

Background: We investigated the effect of root extracts from the traditional Chinese medicine (TCM) plants Glycyrrhiza glabra L., Paeonia lactiflora Pall., and the leaf extract of Eriobotrya japonica (Thunb.) Lindl., and their six major secondary metabolites, glycyrrhizic acid, 18β glycyrrhetinic acid, liquiritigenin, isoliquiritigenin, paeoniflorin, and ursolic acid, on lipopolysaccharide (LPS)-induced NF-κB expression and NF-κB-regulated pro-inflammatory factors in murine macrophage RAW 264.7 cells. Methods: The cytotoxicity of the substances was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. RAW 264.7 cells were treated with LPS (1 μg/mL) or LPS plus single substances; the gene expression levels of NF-κB subunits (RelA, RelB, c-Rel, NF-κB1, and NF-κB2), and of ICAM-1, TNF-α, iNOS, and COX-2 were measured employing real-time PCR; nitric oxide (NO) production by the cells was quantified with the Griess assay; nuclear translocation of NF-κB was visualized by immunofluorescence microscopy with NF-κB (p65) staining. Results: All the substances showed moderate cytotoxicity against RAW 264.7 cells except paeoniflorin with an IC50 above 1000 μM. Glycyrrhiza glabra extract and Eriobotrya japonica extract, as well as 18β glycyrrhetinic acid and isoliquiritigenin at low concentrations, inhibited NO production in a dose-dependent manner. LPS upregulated gene expressions of NF-κB subunits and of ICAM-1, TNF-α, iNOS, and COX-2 within 8 h, which could be decreased by 18β glycyrrhetinic acid, isoliquiritigenin and ursolic acid similarly to the anti-inflammatory drug dexamethasone. NF-κB translocation from cytoplasm to nucleus was observed after LPS stimulation for 2 h and was attenuated by extracts of Glycyrrhiza glabra and Eriobotrya japonica, as well as by 18β glycyrrhetinic acid, isoliquiritigenin, and ursolic acid. Conclusions: 18β glycyrrhetinic acid, isoliquiritigenin, and ursolic acid inhibited the gene expressions of ICAM-1, TNF-α, COX-2, and iNOS, partly through inhibiting NF-κB expression and attenuating NF-κB nuclear translocation. These substances showed anti-inflammatory activity. Further studies are needed to elucidate the exact mechanisms and to assess their usefulness in therapy.

MicroRNA-365a-3p inhibits c-Rel-mediated NF-κB signaling and the progression of pancreatic cancer.

NF-κB contributes to the aggressiveness of pancreatic ductal adenocarcinoma (PDA), which is counteracted by the bioactive agent sulforaphane. We investigated sulforaphane-induced microRNA signaling and its influence on progression features. Using established cell lines, microRNA and gene arrays, we predicted miR-365a as the top candidate for the sulforaphane-induced inhibition of the NF-κB subunit c-Rel. The lipofection of miR-365a-3p mimics inhibited the luciferase activity of a c-Rel 3'-UTR construct, as well as c-Rel expression, NF-κB activity, and tumor viability, migration, and clonogenicity, whereas apoptosis was induced. In vivo, miR-365a-3p reduced the volume of tumor xenografts and the expression of progression markers. In a tissue array, the expression of miR-365a-3p was absent in almost all 91 malignant tissues but not in 5 normal tissues, thus confirming the previous results. Our observations suggest that sulforaphane-induced miR-365a-3p expression inhibits NF-κB activity by downregulating c-Rel, which prevents the progression of PDA.

A cytokine network involving IL-36γ, IL-23, and IL-22 promotes antimicrobial defense and recovery from intestinal barrier damage

The gut epithelium acts to separate host immune cells from unrestricted interactions with the microbiota and other environmental stimuli. In response to epithelial damage or dysfunction, immune cells are activated to produce interleukin (IL)-22, which is involved in repair and protection of barrier surfaces. However, the specific pathways leading to IL-22 and associated antimicrobial peptide (AMP) production in response to intestinal tissue damage remain incompletely understood. Here, we define a critical IL-36/IL-23/IL-22 cytokine network that is instrumental for AMP production and host defense. Using a murine model of intestinal damage and repair, we show that IL-36γ is a potent inducer of IL-23 both in vitro and in vivo. IL-36γ-induced IL-23 required Notch2-dependent (CD11b+CD103+) dendritic cells (DCs), but not Batf3-dependent (CD11b-CD103+) DCs or CSF1R-dependent macrophages. The intracellular signaling cascade linking IL-36 receptor (IL-36R) to IL-23 production by DCs involved MyD88 and the NF-κB subunits c-Rel and p50. Consistent with in vitro observations, IL-36R- and IL-36γ-deficient mice exhibited dramatically reduced IL-23, IL-22, and AMP levels, and consequently failed to recover from acute intestinal damage. Interestingly, impaired recovery of mice deficient in IL-36R or IL-36γ could be rescued by treatment with exogenous IL-23. This recovery was accompanied by a restoration of IL-22 and AMP expression in the colon. Collectively, these data define a cytokine network involving IL-36γ, IL-23, and IL-22 that is activated in response to intestinal barrier damage and involved in providing critical host defense.

Driving Rel-iant Tregs toward an Identity Crisis

Inhibiting Treg cell function in tumors is an attractive strategy to improve anti-cancer immunity. In a pair ofpapers in Immunity and Cell, Ghosh and colleagues show that the canonical NF-κB subunits p65 and c-Rel have non-redundant, critical roles in promoting Treg cell development and function (Oh etal., 2017). Targeting c-Rel blunts Treg cell immunosuppressive activity in the tumor microenvironment and enhances anti-tumor Tcell responses (Grinberg-Bleyer etal., 2017).

CRISPR/Cas9-mediated knockout of c-REL in HeLa cells results in profound defects of the cell cycle

Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit c-REL in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the c-REL gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of c-REL to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of c-REL-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to c-REL+/+ HeLa Kyoto cells. c-REL-/- cells further showed significantly decreased expression levels of c-REL target genes in comparison to wt. In accordance to our proliferation data, we observed the c-REL knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

Central immune tolerance depends on crosstalk between the classical and alternative NF-κB pathways in medullary thymic epithelial cells

Medullary thymic epithelial cells (mTECs) contribute to self-tolerance by expressing and presenting peripheral tissue antigens for negative selection of autoreactive T cells and differentiation of natural regulatory T cells. The molecular control of mTEC development remains incompletely understood. We here demonstrate by TEC-specific gene manipulation in mice that the NF-κB transcription factor subunit RelB, which is activated by the alternative NF-κB pathway, regulates development of mature mTECs in a dose-dependent manner. Mice with conditional deletion of Relb lacked mature mTECs and developed spontaneous autoimmunity. In addition, the NF-κB subunits RelA and c-Rel, which are both activated by classical NF-κB signaling, were jointly required for mTEC differentiation by directly regulating the transcription of Relb. Our data reveal a crosstalk mechanism between classical and alternative NF-κB pathways that tightly controls the development of mature mTECs to ensure self-tolerance.