Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) Signaling

Excessive nuclear factor κB (NF-κB) activation should be precisely controlled as it contributes to multiple immune and inflammatory diseases. However, the negative regulatory mechanisms of NF-κB activation still need to be elucidated. Various types of polyubiquitin chains have proved to be involved in the process of NF-κB activation. Many negative regulators linked to ubiquitination, such as A20 and CYLD, inhibit IκB kinase activation in the NF-κB signaling pathway. To find new NF-κB signaling regulators linked to ubiquitination, we used a small scale siRNA library against 51 ubiquitin-associated domain-containing proteins and screened out UBXN1, which contained both ubiquitin-associated and ubiquitin regulatory X (UBX) domains as a negative regulator of TNFα-triggered NF-κB activation. Overexpression of UBXN1 inhibited TNFα-triggered NF-κB activation, although knockdown of UBXN1 had the opposite effect. UBX domain-containing proteins usually act as valosin-containing protein (VCP)/p97 cofactors. However, knockdown of VCP/p97 barely affected UBXN1-mediated NF-κB inhibition. At the same time, we found that UBXN1 interacted with cellular inhibitors of apoptosis proteins (cIAPs), E3 ubiquitin ligases of RIP1 in the TNFα receptor complex. UBXN1 competitively bound to cIAP1, blocked cIAP1 recruitment to TNFR1, and sequentially inhibited RIP1 polyubiquitination in response to TNFα. Therefore, our findings demonstrate that UBXN1 is an important negative regulator of the TNFα-triggered NF-κB signaling pathway by mediating cIAP recruitment independent of VCP/p97.     

TRIM22 activates NF-κB signaling in glioblastoma by accelerating the degradation of IκBα

NF-κB signaling plays a critical role in tumor growth and treatment resistance in GBM as in many other cancers. However, the molecular mechanisms underlying high, constitutive NF-κB activity in GBM remains to be elucidated. Here, we screened a panel of tripartite motif (TRIM) family proteins and identified TRIM22 as a potential activator of NF-κB using an NF-κB driven luciferase reporter construct in GBM cell lines. Knockout of TRIM22 using Cas9-sgRNAs led to reduced GBM cell proliferation, while TRIM22 overexpression enhanced proliferation of cell populations, in vitro and in an orthotopic xenograft model. However, two TRIM22 mutants, one with a critical RING-finger domain deletion and the other with amino acid changes at two active sites of RING E3 ligase (C15/18A), were both unable to promote GBM cell proliferation over controls, thus implicating E3 ligase activity in the growth-promoting properties of TRIM22. Co-immunoprecipitations demonstrated that TRIM22 bound a negative regulator of NF-κB, NF-κB inhibitor alpha (IκBα), and accelerated its degradation by inducing K48-linked ubiquitination. TRIM22 also formed a complex with the NF-κB upstream regulator IKKγ and promoted K63-linked ubiquitination, which led to the phosphorylation of both IKKα/β and IκBα. Expression of a non-phosphorylation mutant, srIκBα, inhibited the growth-promoting properties of TRIM22 in GBM cell lines. Finally, TRIM22 was increased in a cohort of primary GBM samples on a tissue microarray, and high expression of TRIM22 correlated with other clinical parameters associated with progressive gliomas, such as wild-type IDH1 status. In summary, our study revealed that TRIM22 activated NF-κB signaling through posttranslational modification of two critical regulators of NF-κB signaling in GBM cells.Subject terms: CNS cancer, Oncogenes, Ubiquitin ligases     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Negative Regulation of CARD11 Signaling and Lymphoma Cell Survival by the E3 Ubiquitin Ligase RNF181

NF-κB activation downstream of antigen receptor engagement is a highly regulated event required for lymphocyte activation during the adaptive immune response. The pathway is often dysregulated in lymphoma, leading to constitutive NF-κB activity that supports the aberrant proliferation of transformed lymphocytes. To identify novel regulators of antigen receptor signaling to NF-κB, we developed bioluminescence resonance energy transfer-based interaction cloning (BRIC), a screening strategy that can detect protein-protein interactions in live mammalian cells in a high-throughput manner. Using this strategy, we identified the RING finger protein RNF181 as an interactor of CARD11, a key signaling scaffold in the antigen receptor pathway. We present evidence that RNF181 functions as an E3 ubiquitin ligase to inhibit antigen receptor signaling to NF-κB downstream of CARD11. The levels of the obligate signaling protein Bcl10 are reduced by RNF181 even prior to signaling, and Bcl10 can serve as a substrate for RNF181 E3 ligase activity in vitro. Furthermore, RNF181 limits the proliferation of human diffuse large B cell lymphoma cells that depend upon aberrant CARD11 signaling to NF-κB for growth and survival in culture. Our results define a new regulatory checkpoint that can modulate the output of CARD11 signaling to NF-κB in both normal and transformed lymphocytes.     

Cell shape and the microenvironment regulate nuclear translocation of NF-κB in breast epithelial and tumor cells

Although a great deal is known about the signaling events that promote nuclear translocation of NF-κB, how cellular biophysics and the microenvironment might regulate the dynamics of this pathway is poorly understood. In this study, we used high-content image analysis and Bayesian network modeling to ask whether cell shape and context features influence NF-κB activation using the inherent variability present in unperturbed populations of breast tumor and non-tumor cell lines. Cell–cell contact, cell and nuclear area, and protrusiveness all contributed to variability in NF-κB localization in the absence and presence of TNFα. Higher levels of nuclear NF-κB were associated with mesenchymal-like versus epithelial-like morphologies, and RhoA-ROCK-myosin II signaling was critical for mediating shape-based differences in NF-κB localization and oscillations. Thus, mechanical factors such as cell shape and the microenvironment can influence NF-κB signaling and may in part explain how different phenotypic outcomes can arise from the same chemical cues.     

TRIM Protein-Mediated Regulation of Inflammatory and Innate Immune Signaling and Its Association with Antiretroviral Activity

Members of the tripartite interaction motif (TRIM) family of E3 ligases are emerging as critical regulators of innate immunity. To identify new regulators, we carried out a screen of 43 human TRIM proteins for the ability to activate NF-κB, AP-1, and interferon, hallmarks of many innate immune signaling pathways. We identified 16 TRIM proteins that induced NF-κB and/or AP-1. We found that one of these, TRIM62, functions in the TRIF branch of the TLR4 signaling pathway. Knockdown of TRIM62 in primary macrophages led to a defect in TRIF-mediated late NF-κB, AP-1, and interferon production after lipopolysaccharide challenge. We also discovered a role for TRIM15 in the RIG-I-mediated interferon pathway upstream of MAVS. Knockdown of TRIM15 limited virus/RIG-I ligand-induced interferon production and enhanced vesicular stomatitis virus replication. In addition, most TRIM proteins previously identified to inhibit murine leukemia virus (MLV) demonstrated an ability to induce NF-κB/AP-1. Interfering with the NF-κB and AP-1 signaling induced by the antiretroviral TRIM1 and TRIM62 proteins rescued MLV release. In contrast, human immunodeficiency virus type 1 (HIV-1) gene expression was increased by TRIM proteins that induce NF-κB. HIV-1 resistance to inflammatory TRIM proteins mapped to the NF-κB sites in the HIV-1 long terminal repeat (LTR) U3 and could be transferred to MLV. Thus, our work identifies new TRIM proteins involved in innate immune signaling and reinforces the striking ability of HIV-1 to exploit innate immune signaling for the purpose of viral replication.     

An Update on the Multifaceted Role of NF-kappaB in Endometriosis

Endometriosis remains a common but challenging gynecological disease among reproductive-aged women with an unclear pathogenesis and limited therapeutic options. Numerous pieces of evidence suggest that NF-κB signaling, a major regulator of inflammatory responses, is overactive in endometriotic lesions and contributes to the onset, progression, and recurrence of endometriosis. Several factors, such as estrogen, progesterone, oxidative stress, and noncoding RNAs, can regulate NF-κB signaling in endometriosis. In the present review, we discuss the mechanisms by which these factors regulate NF-κB during endometriosis progression and provide an update on the role of NF-κB in affecting endometriotic cells, peritoneal macrophages (PMs) as well as endometriosis-related symptoms, such as pain and infertility. Furthermore, the preclinical drugs for blocking NF-κB signaling in endometriosis are summarized, including plant-derived medicines, NF-κB inhibitors, other known drugs, and the potential anti-NF-κB drugs predicted through the Drug-Gene Interaction Database. The present review discusses most of the studies concerning the multifaceted role of NF-κB signaling in endometriosis and provides a summary of NF-κB-targeted treatment in detail.