Degradation Of TRAF6 Research Articles

ASB3 expression aggravates inflammatory bowel disease by targeting TRAF6 protein stability and affecting the intestinal microbiota.

Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

ZIKV induces P62-mediated autophagic degradation of TRAF6 through TRAF6-NS1 interaction

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is crucial in flavivirus infections, modulating the host immune response through interactions with viral proteins. Despite its importance, the relationship between TRAF6 and Zika virus (ZIKV) remains poorly understood. Our prior proteomics analysis revealed reduced TRAF6 protein levels in ZIKV-infected human trophoblast cells compared to non-infected controls. Subsequent studies in cell models and murine tissues confirmed a significant reduction in both TRAF6 mRNA and protein levels post-ZIKV infection. Further investigations unveiled that ZIKV induces P62-mediated degradation of TRAF6, with NS1 identified as the primary contributor. Co-localization and interaction studies demonstrated that NS1 promotes the association of P62, a key autophagy mediator, with TRAF6. Notably, our findings revealed TRAF6 enhances ZIKV infection, NS1 ubiquitination, NS1 expression, and the production of inflammatory cytokines and chemokines. These insights highlight the intricate TRAF6-ZIKV relationship, offering potential for drug targeting NS1-TRAF6 interactions to manage ZIKV infections effectively.

Abstract Mo020: Loss of TRAF2 Signaling Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy

Rationale: Cytokines such as TNFα have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). While TNFα can elicit different cellular responses including survival or death, the mechanisms underlying these divergent outcomes in the heart remains cryptic. The E3 ubiquitin ligase TRAF2 provides a critical signaling platform for K63 - linked ubiquitination of RIPK1, crucial for NF-kB activation by TNFα. Whether alterations in TNFα-TRAF2 signaling underlie the cardiotoxic effects of DOX, remains poorly understood. Results: In contrast to vehicle treated mice, severe ultrastructural defects including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFa levels were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFa in DOX cardiotoxicity, we discovered that in the absence of DOX, NF-kB was readily activated by TNFa. However, TNFα -mediated NF-kB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked poly-ubiquitination of RIPK1, attributed to the proteasomal degradation of TRAF2. Further, TRAF2 protein expression was markedly reduced in hearts of cancer patients treated with DOX. Impaired TRAF2 signaling resulted in the activation of Bnip3 and mitochondrial perturbations, including disrupted bioenergetics, loss of membrane potential and permeability transition pore opening. We further established that the reciprocal actions of the ubiquitinating and de-ubiquitinating enzymes c-IAP1 and USP19 respectively regulated the proteasomal degradation of TRAF2 in DOX treated cardiac myocytes. Importantly, an E3 ligase mutant of c-IAP1(c-IAP1 H588A) or gain of function of USP19, prevented proteasomal degradation of TRAF2 and DOX -induced cell death. Further, wild type TRAF2 but not a RING finger mutant defective for K63 ubiquitination of RIPK1, restored NF-kB signaling and suppressed DOX-induced cardiac cell death. Finally, cardiomyocyte-restricted expression of TRAF2 (AAV9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Conclusions: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX, sensitized cardiac myocytes to TNFa mediated necrotic cell death.

Active fraction of Polyrhachis vicina (Rogers) inhibits osteoclastogenesis by targeting Trim38 mediated proteasomal degradation of TRAF6

BackgroundReactive Oxygen Species (ROS) is a key factor in the pathogenesis of osteoporosis (OP) primarily characterized by excessive osteoclast activity. Active fraction of Polyrhachis vicina Rogers (AFPR) exerts antioxidant effects and possesses extensive promising therapeutic effects in various conditions, however, its function in osteoclastogenesis and OP is unknown. PurposeThe aim of this study is to elucidate the cellular and molecular mechanisms of AFPR in OP. Study design and methodsCCK8 assay was used to evaluate the cell viability under AFPR treatment. TRAcP staining, podosome belts staining and bone resorption were used to test the effect of AFPR on osteoclastogenesis. Immunofluorescence staining was used to observe the effect of AFPR on ROS production. si-RNA transfection, coimmunoprecipitation and Western-blot were used to clarify the underlying mechanisms. Further, an ovariectomy (OVX) -induced OP mice model was used to identify the effect of AFPR on bone loss using Micro-CT scanning and histological examination. ResultsIn the present study, AFPR inhibited osteoclast differentiation and bone resorption induced by nuclear factor-κB receptor activator (NF-κB) ligand (RANKL) in dose-/ time-dependent with no cytotoxicity. Meanwhile, AFPR decreased RANKL-mediated ROS levels and enhanced ROS scavenging enzymes. Mechanistically, AFPR promoted proteasomal degradation of TRAF6 by significantly upregulating its K48-linked ubiquitination, subsequently inhibiting NFATc1 activity. We further observed that tripartite motif protein 38 (TRIM38) could mediate the ubiquitination of TRAF6 in response to RANKL. Moreover, TRIM38 could negatively regulate the RANKL pathway by binding to TRAF6 and promoting K48-linked polyubiquitination. In addition, TRIM38 deficiency rescued the inhibition of AFPR on ROS and NFATc1 activity and osteoclastogenesis. In line with these results, AFPR reduced OP caused by OVX through ameliorating osteoclastogenesis. ConclusionAFPR alleviates ovariectomized-induced bone loss via suppressing ROS and NFATc1 by targeting Trim38 mediated proteasomal degradation of TRAF6. The research offers innovative perspectives on AFPR's suppressive impact in vivo OVX mouse model and in vitro, and clarifies the fundamental mechanism.

14-3-3ζ suppresses RANKL signaling by destabilizing TRAF6

Macrophages are essential regulators of inflammation and bone loss. RANKL, a pro-inflammatory cytokine, is responsible for macrophage differentiation to osteoclasts and bone loss. We recently showed that 14-3-3ζ-knockout (YwhazKO) rats exhibit increased bone loss in the inflammatory arthritis model. 14-3-3ζ is a cytosolic adaptor protein that actively participates in many signaling transductions. However, the role of 14-3-3ζ in RANKL signaling or bone remodeling is unknown. We investigated how 14-3-3ζ affects osteoclast activity by evaluating its role in RANKL signaling. We utilized 14-3-3ζ-deficient primary bone marrow-derived macrophages (BMDMs) obtained from wildtype (Wt) and YwhazKO animals, and RAW cells generated using CRISPR-Cas9. Our results showed that 14-3-3ζ-deficient macrophages, upon RANKL stimulation, have bigger and stronger TRAP-positive multinucleated cells and increased bone resorption activity. The presence of 14-3-3ζ suppressed RANKL-induced MAPK and AKT phosphorylation, transcription factors (NFATC1 and p65) nuclear translocation, and subsequently, gene induction (Rank, Acp5, and Ctsk). Mechanistically, 14-3-3ζ interacts with TRAF6, an essential component of the RANKL receptor complex. Upon RANKL stimulation, 14-3-3ζ-TRAF6 interaction was increased, while RANK-TRAF6 interaction was decreased. Importantly, 14-3-3ζ supported TRAF6 ubiquitination and degradation by the proteasomal pathway, thus dampening the downstream RANKL signaling. Together, we show that 14-3-3ζ regulates TRAF6 levels to suppress inflammatory RANKL signaling and osteoclast activity. To the best of our knowledge, this is the first report on 14-3-3ζ regulation of RANKL signaling and osteoclast activation.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor β (TGFβ)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFβ and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFβ causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3β/β-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFβ+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

CircMIB2 therapy can effectively treat pathogenic infection by encoding a novel protein

The mRNA therapy is widely used in the treatment of diseases due to its efficient characteristics, and the COVID-19 vaccine is the application of mRNA therapy. However, due to the instability of mRNA, mRNA vaccines often need lots of modifications to ensure its stability. Recent research shows that circRNA with stable RNA structure can encode protein, which provides a new direction for mRNA therapy. Here, we discovered a novel circRNA (circMIB2) derived from E3 ubiquitin-protein ligase MIB2 (MIB2) gene in lower vertebrate fish, which can translate into a 134 amino acid protein (MIB2-134aa) through m6A modification, and is involved in innate immunity. MIB2-134aa is completely consistent with the amino acid sequence of the two domains of host gene MIB2 protein; host gene MIB2 can target TRAF6 through the two domains and inhibit the innate immune response by promoting the ubiquitination degradation of the K11-link of TRAF6, MIB2-134aa also targets TRAF6 through these same domains. Interestingly, MIB2-134aa greatly reduced the degradation of TRAF6 by its host gene MIB2. More importantly, we found that circRNA therapy of circMIB2 can significantly inhibit the colonization of Vibrio anguillarum in zebrafish, and it provides a new direction for the treatment of pathogenic diseases of fish.

Heterogeneous Nuclear Protein U Degraded the m6A Methylated TRAF3 Transcript by YTHDF2 To Promote Porcine Epidemic Diarrhea Virus Replication.

Porcine epidemic diarrhea virus (PEDV) belongs to the genus Alphacoronavirus of the Coronaviridae family and can cause fatal watery diarrhea in piglets, causing significant economic losses. Heterogeneous nuclear protein U (HNRNPU) is a novel RNA sensor involved in sensing viral RNA in the nucleus and mediating antiviral immunity. However, it remains elusive whether and how cytoplasmic PEDV can be sensed by the RNA sensor HNRNPU. In this study we determined that HNRNPU was the binding partner of Nsp13 by immunoprecipitation-liquid chromatography-tandem mass spectrometry (IP/LC-MS/MS) analysis. The interaction between Nsp13 and HNRNPU was demonstrated by using coimmunoprecipitation and confocal immunofluorescence. Next, we identified that HNRNPU expression is significantly increased during PEDV infection, whereas the transcription factor hepatocyte nuclear factor 1α (HNF1A) could negatively regulate HNRNPU expression. HNRNPU was retained in the cytoplasm by interaction with PEDV Nsp13. We found that HNRNPU overexpression effectively facilitated PEDV replication, while knockdown of HNRNPU impaired viral replication, suggesting a promoting function of HNRNPU to PEDV infection. Additionally, HNRNPU was found to promote PEDV replication by affecting TRAF3 degradation at the transcriptional level to inhibit PEDV-induced beta interferon (IFN-β) production. Mechanistically, HNRNPU downregulates TRAF3 mRNA levels via the METTL3-METTL14/YTHDF2 axis and regulates immune responses through YTHDF2-dependent mRNA decay. Together, our findings reveal that HNRNPU serves as a negative regulator of innate immunity by degrading TRAF3 mRNA in a YTHDF2-dependent manner and consequently facilitating PEDV propagation. Our findings provide new insights into the immune escape of PEDV. IMPORTANCE PEDV, a highly infectious enteric coronavirus, has spread rapidly worldwide and caused severe economic losses. During virus infection, the host regulates innate immunity to inhibit virus infection. However, PEDV has evolved a variety of different strategies to suppress host IFN-mediated antiviral responses. Here, we identified that HNRNPU interacted with viral protein Nsp13. HNRNPU protein expression was upregulated, and the transcription factor HNF1A could negatively regulate HNRNPU expression during PEDV infection. HNRNPU also downregulated TRAF3 mRNA through the METTL3-METTL14/YTHDF2 axis to inhibit the production of IFN-β and downstream antiviral genes in PEDV-infected cells, thereby promoting viral replication. Our findings reveal a new mechanism with which PEDV suppresses the host antiviral response.

Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.

Anti-apoptotic Splicing Variant of AIMP2 Recover Mutant SOD1-Induced Neuronal Cell Death.

Although a couple of studies have reported that mutant superoxide dismutase 1 (SOD1), one of the causative genes of familial amyotrophic lateral, interacts physically with lysyl-tRNA synthetase (KARS1) by a gain of function, there is limited evidence regarding the detailed mechanism about how the interaction leads to neuronal cell death. Our results indicated that the aminoacyl-tRNA synthetase-interacting multi-functional protein 2 (AIMP2) mediated cell death upon the interplay between mutant SOD1 and KARS1 in ALS. Binding of mutant SOD1 with KARS1 led to the release of AIMP2 from its original binding partner KARS1, and the free form of AIMP2 induced TRAF2 degradation followed by TNF-α-induced cell death. We also suggest a therapeutic application that overexpression of DX2, the exon 2-deleted antagonistic splicing variant of AIMP2 (AIMP2-DX2), reduced neuronal cell death in the ALS mouse model. Expression of DX2 suppressed TRAF2 degradation and TNF-α-induced cell death by competing mode of action against full-length AIMP2. Motor neuron differentiated form iPSC showed a resistance in neuronal cell death after DX2 administration. Further, intrathecal administration of DX2-coding adeno-associated virus (AAV) improved locomotive activity and survival in a mutant SOD1-induced ALS mouse model. Taken together, these results indicated that DX2 could prolong life span and delay the ALS symptoms through compensation in neuronal inflammation.

Minimal structure of IRAK-1 to induce degradation of TRAF6

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1–102), a serine/proline/threonine-rich ProST domain (amino acids 103–197), a central kinase domain with an activation loop (amino acids 198–522), and the C-terminal C1 and C2 domains (amino acids 523–712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585–591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants–including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis–induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis–stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre–mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis–induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection–induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis–induced inflammatory immune responses and mapped out a novel SGK1–TRAF2/3–c-Jun–NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.

HnRNPub inhibits LPS-induced NF-κB pathway by targeting TRAF6 for K48-linked ubiquitination in miiuy croaker (Miichthys miiuy)

As an important adaptor protein in innate immunity, TRAF6 is not only responsible for the transduction of signal pathways, but its E3 ligase activity to transfer ubiquitination has also been widely studied. Under LPS stimulation, TRAF6 transfers the K63-linked ubiquitination chain to TAK1, which in turn activates the transcription factor NF-κB and cell signaling factors downstream of the signaling pathway. However, how TRAF6 expression is regulated remains largely unknown, especially in teleost. In this study, we identified hnRNPub as a suppressor of TRAF6 expression. The mRNA level of hnRNPub significantly increased under LPS stimulation, and hnRNPub inhibited NF-κB signaling pathway by targeting TRAF6. Knockdown of hnRNPub potentiated inflammatory cytokines, such as TNFα,IL-1β,IL-8. Mechanistically, hnRNPub inhibited NF-κB signaling pathway through mediating K48-linked ubiquitination and proteasomal degradation of TRAF6. Thus, our findings reveal that hnRNPub limits LPS-induced innate activation by promoting K48-linked polyubiquitination and proteasomal degradation of TRAF6.

The small RNA mascRNA differentially regulates TLR-induced proinflammatory and antiviral responses.

MALAT1-associated small cytoplasmic RNA (mascRNA) is a highly conserved transfer RNA–like (tRNA-like) noncoding RNA whose function remains largely unknown. We show here that this small RNA molecule played a role in the stringent control of TLR-mediated innate immune responses. mascRNA inhibited activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling and the production of inflammatory cytokines in macrophages stimulated with LPS, a TLR4 ligand. Furthermore, exogenous mascRNA alleviated LPS-induced lung inflammation. However, mascRNA potentiated the phosphorylation of IRF3 and STAT1 and the transcription of IFN-related genes in response to the TLR3 ligand poly(I:C) both in vitro and in vivo. Mechanistically, mascRNA was found to enhance K48-linked ubiquitination and proteasomal degradation of TRAF6, thereby negatively regulating TLR-mediated MyD88-dependent proinflammatory signaling while positively regulating TRIF-dependent IFN signaling. Additionally, heterogeneous nuclear ribonucleoprotein H (hnRNP H) and hnRNP F were found to interact with mascRNA, promote its degradation, and contribute to the fine-tuning of TLR-triggered immune responses. Taken together, our data identify a dual role of mascRNA in both negative and positive regulation of innate immune responses.

BAFF signaling in health and disease.

BAFF is a critical cytokine supporting the survival of mature naïve B cells, acting through the BAFFR receptor. Recent studies show that BAFF and BAFFR are also required for the survival of memory B cells, autoimmune B cells as well as malignant chronic lymphocytic leukaemia (CLL) cells. BAFFR cooperates with other receptors, notably the B cell antigen receptor (BCR), a process which is critical for the expansion of autoimmune and CLL cells. This crosstalk may be mediated by TRAF3 which interacts with BAFFR and with CD79A, a signalling subunit of the BCR and the downstream SYK kinase, inhibiting its activity. BAFF binding to BAFFR leads to degradation of TRAF3 which may relieve inhibition of SYK activity transducing signals to pathways required for B cell survival. BAFFR activates both canonical and non-canonical NF-κB signalling and both pathways play important roles in the survival of B cells and CLL cells.

EI24 alleviates renal interstitial fibrosis through inhibition of epithelial‐mesenchymal transition and fibroblast activation

Etoposide-induced 2.4 (EI24) exerts tumor suppressor activity through participating in cell apoptosis, autophagy, and inflammation. However, its role in renal diseases has not been elucidated. This study showed that the EI24 level decreased gradually in the kidneys of mice with unilateral ureteral obstruction (UUO) and in another fibrosis model induced by diabetic kidney disease. The overexpression of EI24 was used to investigate the possible role both in vivo and in vitro. The overexpression 1day after UUO through tail vein injection alleviated the progression of renal interstitial fibrosis (RIF). EI24 inhibited epithelial-mesenchymal transition, excessive deposition of the extracellular matrix, and activation of fibroblasts. Furthermore, administration of EI24-overexpressing plasmids restrained the phosphorylation of nuclear factor-κB (NF-κB) and c-Jun kinase (JNK) through regulating the proteasome-dependent degradation of TRAF2, and then, inhibited the expression of downstream inflammation-associated cytokines (interleukin-6, tumor necrosis factor-α, and monocyte chemotactic protein-1) and infiltration of macrophages and neutrophils in mouse kidney after UUO. In conclusion, the data indicated that EI24, a novel anti-fibrosis regulator, was important in the progression of RIF.

An Antioxidant Sesquiterpene Inhibits Osteoclastogenesis Via Blocking IPMK/TRAF6 and Counteracts OVX-Induced Osteoporosis in Mice.

Excessive bone resorption induced by increased osteoclast activity in postmenopausal women often causes osteoporosis. Although the pharmacological treatment of osteoporosis has been extensively developed, a safer and more effective treatment is still needed. Here, we found that curcumenol (CUL), an antioxidant sesquiterpene isolated from Curcuma zedoaria, impaired receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in vitro, whereas the osteoblastogenesis of MC3T3-E1 cells was not affected. We further demonstrated that CUL treatment during RANKL-induced osteoclastogenesis promotes proteasomal degradation of TRAF6 by increasing its K48-linked polyubiquitination, leading to suppression of mitogen-activated protein kinases (MAPKs) and NF-κB pathways and the production of reactive oxygen species (ROS). We also showed that inositol polyphosphate multikinase (IPMK) binds with TRAF6 to reduce its K48-linked polyubiquitination under RANKL stimulation. Concurrently, IPMK deficiency inhibits osteoclast differentiation. The binding between IPMK and TRAF6 blocked by CUL treatment was found in our study. Finally, we confirmed that CUL treatment prevented ovariectomy (OVX)-induced bone loss in mice. In summary, our study demonstrates that CUL could impair the stability of TRAF6 enhanced by IPMK and suppress excessive osteoclast activity in estrogen-deficient mice to treat osteoporosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Rictor Is a Novel Regulator of TRAF6/TRAF3 in Osteoclasts.

Tumor necrosis factor receptor-associated factors (TRAFs) are crucial for receptor activator of nuclear factor-κB (RANK) activation in osteoclasts. However, the upstream mechanisms of TRAF members in the osteoclastic lineage remain largely unknown. Here, we demonstrated that Rictor, a key component of mechanistic target of rapamycin complex 2 (mTORC2), was crucial for TRAF6/TRAF3 expression in osteoclasts. Our ex vivo and in vivo studies showed that Rictor ablation from the osteoclastic lineage reduced osteoclast numbers and increased bone mass in mice. Mechanistically, we found that Rictor ablation restricted osteoclast formation, which disrupted TRAF6 stability and caused autophagy block in a manner distinct from mTORC1, resulting in reduced TRAF3 degradation. Boosting TRAF6 expression or knockdown of TRAF3 levels in Rictor-deficient cells could both overcome the defect. Moreover, Rictor could interact with TRAF6 upon RANK ligand (RANKL) stimulation and loss of Rictor impaired TRAF6 stability and promoted its ubiquitinated degradation. These findings established an innovative link between Rictor, TRAF protein levels, and autophagic block. More importantly, mTOR complexes in the osteoclastic lineage are likely switches for coordinating TRAF6 and TRAF3 protein levels, and Rictor may function as an essential upstream regulator of TRAF6/TRAF3 that is partially independent of mTORC1 activity. Inhibitors targeting Rictor may therefore be valuable for preventing or treating osteoclast-related diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).

Artesunate inhibits Sjögren's syndrome-like autoimmune responses and BAFF-induced B cell hyperactivation via TRAF6-mediated NF-κB signaling

BackgroundHyperactivation of B cells by activators has been demonstrated to play a central role in the pathogenesis of Sjögren's syndrome (SS). In this study, we found that artesunate (ART) can attenuate BAFF-induced B cell hyperactivation and SS-like symptoms in NOD/Ltj mice. PurposeTo determine the efficacy of ART in attenuating SS-like symptoms in vivo and explore the underlying mechanism in vitro. Study designART was intragastrically injected into SS-like NOD/Ltj mice. The cytokine hsBAFF was used to activate Raji and Daudi B cells to mimic B cell hyperactivation in vitro. MethodsThe efficacy of ART in inhibiting SS progression was studied in NOD/Ltj mice. Salivary flow rate, the number of lymphocytic infiltration foci, the level of autoantibodies and the extent of B cell infiltration were measured in the indicated groups. CCK-8 assays, flow cytometry-based EdU staining and Annexin V/PI staining were also used to detect the effect of ART on the survival and proliferation mechanism in BAFF-induced Raji and Daudi cells. Further studies determined that TRAF6 degradation is a potential mechanism by which ART determines B cell fate. ResultsTreatment with ART inhibited lymphocytic foci formation, B cell infiltration and autoantibody secretion in SS-like NOD/Ltj mice. In vitro assay results indicated that ART effectively inhibited BAFF-induced viability, survival and proliferation of neoplastic B cells. Mechanistically, ART targeted BAFF-activated NFκB by regulating the proteasome-mediated degradation of TRAF6 in Raji and Daudi cells. ConclusionART ameliorated murine SS-like symptoms and regulated TRAF6-NFκB signaling, thus determining survival and proliferation of B cells.