TRAF Proteins Research Articles

LncRNA MALAT1 promotes high glucose-induced inflammatory response of microglial cells via provoking MyD88/IRAK1/TRAF6 signaling

Although a large number of studies have confirmed from multiple levels that diabetes mellitus (DM) promotes cerebral ischemic reperfusion (I/R) injury, but the precise mechanism is still unclear. A cerebral I/R injury model in diabetic rats was established. The neurological deficit scores and brain edema were monitored at 24 and 72 hours after injury. The peri-infarct cortical tissues of rats were isolated for molecular biology detection. The rat primary microglia and microglia line HAPI were cultured to establish the cell model of DM-I/R by high glucose (HG) and hypoxia-reoxygenation (H/R). The endogenous expression of MALAT1 and MyD88 was regulated by the transfection with pcDNA-MALAT1, si-MALAT1 and si-MyD88, respectively. The cerebral I/R injury model in diabetic rats had more severe neuronal injury as shown by the significantly higher neurological deficit scores and an obvious increasing brain edema at 24 and 72 hours after injury. Moreover, the microglia were activated and induced a large number of inflammatory cytokines TNF-α, IL-1β and IL-6 in the peri-infarct cortical tissues during cerebral I/R injury associated with DM. The expression of MALAT1, MyD88, IRAK1 and TRAF6 protein were significantly up-regulated by DM-I/R in vitro and in vivo. Furthermore, the HG-H/R-induced MALAT1 promoted the inflammatory response in microglia via MyD88/IRAK1/TRAF6 signaling. Our results suggested that MALAT1 mediated the exacerbation of cerebral I/R injury induced by DM through triggering the inflammatory response in microglia via MyD88 signaling.

TNFRSF19 Inhibits TGFβ Signaling through Interaction with TGFβ Receptor Type I to Promote Tumorigenesis.

Genetic susceptibility underlies the pathogenesis of cancer. We and others have previously identified a novel susceptibility gene TNFRSF19, which encodes an orphan member of the TNF receptor superfamily known to be associated with nasopharyngeal carcinoma (NPC) and lung cancer risk. Here, we show that TNFRSF19 is highly expressed in NPC and is required for cell proliferation and NPC development. However, unlike most of the TNF receptors, TNFRSF19 was not involved in NFκB activation or associated with TRAF proteins. We identified TGFβ receptor type I (TβRI) as a specific binding partner for TNFRSF19. TNFRSF19 bound the kinase domain of TβRI in the cytoplasm, thereby blocking Smad2/3 association with TβRI and subsequent signal transduction. Ectopic expression of TNFRSF19 in normal epithelial cells conferred resistance to the cell-cycle block induced by TGFβ, whereas knockout of TNFRSF19 in NPC cells unleashed a potent TGFβ response characterized by upregulation of Smad2/3 phosphorylation and TGFβ target gene transcription. Furthermore, elevated TNFRSF19 expression correlated with reduced TGFβ activity and poor prognosis in patients with NPC. Our data reveal that gain of function of TNFRSF19 in NPC represents a mechanism by which tumor cells evade the growth-inhibitory action of TGFβ.Significance:TNFRSF19, a susceptibility gene for nasopharyngeal carcinoma and other cancers, functions as a potent inhibitor of the TGFβ signaling pathway.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3469/F1.large.jpg Cancer Res; 78(13); 3469-83. ©2018 AACR.

A viral homologue of IPS-1 Interacts with TRAF3 leading to a diminished antiviral state.

Abstract Kaposi’s Sarcoma (KS) is an angioproliferative disease with symptoms including skin lesions that appear at different sites of the body caused by Kaposi’s Sarcoma Associated Herpesvirus (KSHV). The KSHV genome encodes genes pirated from the human genome that are homologous to cellular proteins that disrupt innate immune pathways. We have found a novel ORF in the KSHV genome that is homologous to the human IPS-1 protein. This viral IPS-1 proteins (vIPS-1) contains the PRO domain of IPS-1 but lacks the CARD and TM domains. We hypothesize that vIPS-1 is able to interact with the cellular TRAF3 protein. Western blot data shows that co-expression of vIPS-1 does not affect RIG-I, decreases the stability of human IPS-1 and TRIF, and increases the stability of TRAF3. Also, vIPS-1 interacts with TRAF3 via coimmunoprecipitation, and by co-expressing vIPS-1 with different TRAF3 mutants we found that vIPS-1 interacts with TRAF3 through the TRAF domain which is specifically required for TRAF3-mediated type I IFN production. Furthermore, our data shows that while expression of IPS-1 is able to inhibit VSV-GFP replication, expression of vIPS-1 significantly enhances the VSV-GFP replication, consistent with our hypothesis that vIPS-1 blocks the cellular immune responses. In conclusion, the KSHV vIPS-1 protein blocks antiviral immunity by interacting with TRAF3 leading to a decrease in antiviral activity.

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK-JAK interaction.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1-JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5-/- CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway.

Neuroprotective effect of a physiological ratio of testosterone and estradiol on corticosterone-induced apoptosis in PC12 cells via Traf6/TAK1 pathway

Clinical and experimental studies suggested that testosterone or estradiol alone supplementation may have antidepressant-like effects. However, the synergistic effect of testosterone/estradiol on the treatment of depression and the underlying mechanism remain largely unknown. Here, we hypothesized that an appropriate dosage of testosterone combined with estradiol may became a new therapeutic strategy in depression. Our results indicated that an appropriate testosterone/estradiol ratio of 10:1 (10−8 mol/L testosterone and 10−9 mol/L estradiol) was found, which has a significant synergistic anti-apoptotic effect on PC12 cells injury stimulated by corticosterone. We then confirmed the synergistic effect of the defined testosterone/estradiol ratio in PC12 cells mainly depend on the inhibitory activation of Traf6/TAK1 signaling pathway. Furthermore, we found that the defined testosterone/estradiol ratio could suppress the expression and ubiquitination of Traf6 protein through AR/ERα-mediated pathways. Finally, endogenous interactions of Traf6 with AR/ERα in PC12 cells were found using co-immunoprecipitation assays, and further verified how the defined testosterone/estradiol ratio plays a synergistic role in injury of PC12 cells. The current study demonstrated that an appropriate testosterone/estradiol ratio has a synergistic effect on PC12 cells injury induced by corticosterone through suppressing the activation of Traf6/TAK1 signaling pathway, suggesting that testosterone/estradiol might synergistically protect against neuronal apoptosis to ameliorate depressive symptoms.

Palmitate-induced Slc2a4/GLUT4 downregulation in L6 muscle cells: evidence of inflammatory and endoplasmic reticulum stress involvement

BackgroundObesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by Slc2a4 gene, in L6 skeletal muscle cells.MethodsL6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed.ResultsChronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, P < 0.01) and its mRNA (18%, P < 0.01) expression. Only acute palmitate treatment increased GRP78 (28%, P < 0.05), PERK (98%, P < 0.01), eIF-2A (35%, P < 0.01), IRE1a (60%, P < 0.05) and TRAF2 (23%, P < 0.05) protein content, and PERK phosphorylation (106%, P < 0.001), but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content (~ 30%, P < 0.05) and NFKB binding activity to Slc2a4 gene promoter (~ 45%, P < 0.05).ConclusionDifferent pathways are activated in acute and chronic palmitate induced-repression of Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.

Participation of Toll‐like Receptor (TLR) 9 in Obesity‐Induced Benign Prostatic Hyperplasia (BPH) in Mice: Implication of Periprostatic Fat

IntroductionClinical and experimental studies indicate a strong correlation between obesity and benign prostatic hyperplasia (BPH)[1]. Recently, prostatic inflammation and innate immune system have been shown to play a pivotal role in BPH pathogenesis/progression [2]. In the present study we explored the presence and participation of TLR9 signaling pathway on obesity‐induced BPH in mice.MethodsUsing prostate from C57BL6/JUnib mice, we performed immunofluorescence analysis for TLR9 localization. We also evaluated the effects of the in vitro TLR9 activation by ODN 2395 on functional (concentration‐response curves) and molecular (MYD88, TRAF6 and COX2 ‐ TLR9 pathway protein expression) protocols. Besides, male C57BL6/JUnib (Wild type) and TLR9 mutant mice were fed for 12 weeks with either a standard chow or high‐fat diet (HFD) to induce obesity. Fasting glucose levels and body, epididymal and periprostatic fat weight were evaluated. Concentration‐response curves to phenylephrine (PE; α1‐adrenoceptor agonist) and potassium chloride (KCl; depolarizing agent), as well as neurogenic contractions produced by electrical‐field stimulation (EFS; 1–32 Hz, 50 V, 10 s) were also obtained.ResultsThe immunofluorescence assay revealed the presence of TLR9 in epithelial layer of the mouse prostate. The in vitro TLR9 activation with ODN 2395 (TLR9 agonist; 5 μM) led to a significant decrease (P&lt;0.05) in prostate smooth muscle (PSM) contractions to PE and EFS along with increases in MYD88, TRAF6 and COX2 protein expression in prostate from C57BL6/JUnib mice. After 12 weeks of HFD, the TLR9 mutant obese mice exhibited the same body weight compared with WT obese group, however, fasting glucose (FG) and epididymal fat (EF) were reduced on TLR9 mutant obese mice. Despite improvements in FG and EF, TLR9 mutant obese group showed increases in prostate weight and PSM reactivity to PE, KCl and EFS compared with WT and TLR9 mutant lean group. Interestingly, the periprostatic fat (PF) showed augmented (P&lt;0.001) in TLR9 mutant lean group compared with WT lean group. Under obesity conditions, an increase in PF was seen in WT obese group, which was further increased in TLR9 mutant obese group.ConclusionDespite amelioration in fasting glucose and reduction in epididymal fat, TLR9 mutant obese mice still exhibit BPH, which can be, at least in part, due to augmented periprostatic fat.Support or Funding InformationFinancial support: FAPESP (2016/20592‐8); NIH (NHLBI 001961)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of miR-146b-5p in TLR4 pathway of glomerular mesangial cells with lupus nephritis.

MicroRNAs (miRNAs) are now recognized as important regulators of gene expression. The aim of the study was to investigate the role of miR-146b-5p in the TLR4 pathway and provide the basis for the treatment of lupus nephritis. The glomerular mesangial cells were cultured in vitro and divided into 3 groups: control group, a group of miR-146b-5p mimic added, and a group of miR-146b-5p inhibitor added. The levels of IL-6 and IL-8 in the cell culture supernatant of the three groups were detected by ELISA. The cell proliferation was detected by MTT. The expressions of MiR-146b-5p and TLR4 pathway-associated factor TRAF6 were detected by RT- PCR. The expression of TRAF6 and IRAK1 protein was detected by Western blot. The overexpression of miR-146b-5p could reduce the level of IL6 and IL8 in cell culture and inhibit glomerular mesangial cell proliferation in some degree. Also, the overexpression of miR-146b-5p could inhibit the expressions of TLR4 pathway-associated factor TRAF6 and IRAK1mRNA, and the expressions of TRAF6 and IRAK1 protein. MiR-146b-5p attenuated the inflammatory response of glomerular mesangial cells by inhibiting the expressions levels of TRAF6 and IRAK1 in lupus nephritis.

TLR/NF-κB independent signaling pathway in TNF-α suppression of diabetic MyD88-knockout mice after Lycium barbarum polysaccharides administration

: To investigate the effect of Lycium barbarum polysaccharides (LBPs) on TLR/NF-κB independent pathway and serum tumor necrosis factor (TNF-α) level in diabetic MyD88-knockout mice. : Diabetes was induced by feeding high-fat/high-sugar diet and injection of low-dose streptozotocin in MyD88-knockout mice. The diabetic mice were randomly divided into model group, positive control group and LBPs group. The expressions of TRAM, TRIF, TRAF6, RIP1 and TNF-α mRNA and proteins in mouse peritoneal macrophages were detected by real-time RT-PCR and Western blotting after LBPs treatment for 3 month. Serum TNF-α was determined with ELISA kit. : Real time RT-PCR showed that compared with model group, the relative expressions of Tram, Trif, Traf6 and Tnf-α mRNA in macrophages of LBPs group were significantly decreased and expression of Rip1 was significantly increased (all P<0.05). Expression of TRAM, TRIF, TRAF6, RIP1 and TNF-α proteins as well as serum TNF-α level had no significant difference between LBPs group and model group (all P>0.05). : LBPs may not inhibit serum TNF-α level through TLR/NF-κB independent pathway.

NLRP11 attenuates Toll-like receptor signalling by targeting TRAF6 for degradation via the ubiquitin ligase RNF19A

The adaptor protein TRAF6 has a central function in Toll-like receptor (TLR) signalling, yet the molecular mechanisms controlling its activity and stability are unclear. Here we show that NLRP11, a primate specific gene, inhibits TLR signalling by targeting TRAF6 for degradation. NLRP11 recruits the ubiquitin ligase RNF19A to catalyze K48-linked ubiquitination of TRAF6 at multiple sites, thereby leading to the degradation of TRAF6. Furthermore, deficiency in either NLRP11 or RNF19A abrogates K48-linked ubiquitination and degradation of TRAF6, which promotes activation of NF-κB and MAPK signalling and increases the production of proinflammatory cytokines. Therefore, our findings identify NLRP11 as a conserved negative regulator of TLR signalling in primate cells and reveal a mechanism by which the NLRP11-RNF19A axis targets TRAF6 for degradation.

Role of microRNA-146a in regulation of fibrosis in orbital fibroblasts from patients with Graves’ orbitopathy

AimTo examine the role of microRNA-146a (miR-146a) in the regulation of fibrosis in an in vitro model of Graves’ orbitopathy (GO).MethodsOrbital fat/connective tissues were harvested from patients with GO and...

NLRP11 disrupts MAVS signalosome to inhibit type I interferon signaling and virus-induced apoptosis.

MAVS signalosome plays an important role in RIG-I-like receptor (RLR)-induced antiviral signaling. Upon the recognition of viral RNAs, RLRs activate MAVS, which further recruits TRAF6 and other signaling proteins to initiate type I interferon (IFN) activation. MAVS signalosome also regulates virus-induced apoptosis to limit viral replication. However, the mechanisms that control the activity of MAVS signalosome are still poorly defined. Here, we report NLRP11, a Nod-like receptor, is induced by type I IFN and translocates to mitochondria to interact with MAVS upon viral infection. Using MAVS as a platform, NLRP11 degrades TRAF6 to attenuate the production of type I IFNs as well as virus-induced apoptosis. Our findings reveal the regulatory role of NLRP11 in antiviral immunity by disrupting MAVS signalosome.

MicroRNA-126 Regulates Inflammatory Cytokine Secretion in Human Gingival Fibroblasts Under High Glucose via Targeting Tumor Necrosis Factor Receptor Associated Factor 6.

MicroRNAs (miRs) play a crucial role in inflammatory diseases, including periodontitis. Meanwhile, miRs act as biomarkers for predicting diabetes mellitus (DM). However, the regulatory mechanism of miR-126 on development of periodontitis in patients with DM still remains unclear. Human gingival fibroblasts were cultured with low (5.5 mmol/L), medium (15 mmol/L), and high (25 mmol/L) glucose, respectively. Expressions of miR-126, tumor necrosis factor (TNF) receptor associated factor (TRAF) 6, and related cytokines were analyzed by real-time polymerase chain reaction (PCR). After transfection with miR-126 mimic, PCR and western blot were performed to detect level of TRAF6, and luciferase reporter assay confirmed if TRAF6 is the direct target of miR-126. Production of cytokines was measured using enzyme-linked immunosorbent assay. Increased glucose significantly suppressed miR-126 expression in human gingival fibroblasts (P <0.05). Also, high glucose increased TRAF6, interleukin (IL)-6, TNF-α, and chemical chemokine ligand (CCL) 2 levels, whereas it decreased IL-10 level. MiR-126 mimic significantly decreased TRAF6 mRNA and protein levels under high glucose (P <0.05). Also, miR-126 directly targeted TRAF6 through binding to its 3' untranslated region in human gingival fibroblasts. Overexpression of miR-126 significantly abrogated high glucose-induced secretion of proinflammatory cytokines such as IL-6, TNF-α, and CCL2 and promoted production of IL-10. These data suggest that miR-126 inhibits inflammation of human gingival fibroblasts under high glucose through targeting TRAF6, which may be a potential therapeutic target for periodontitis concomitant with DM.

A Dithiol Compound Binds to the Zinc Finger Protein TRAF6 and Suppresses Its Ubiquitination.

Despite various inhibitors targeting the zinc center(s) of enzymes, drugs that target zinc fingers have not been examined in detail. We previously developed a dithiol compound named SN-1 that has an inhibitory effect on the function of zinc finger transcription factors, but its mechanism of action has not yet been elucidated. To establish a general principle for new drugs, the details of the action of SN-1 against a zinc finger protein were examined. As a zinc-finger-containing protein, we focused on TRAF6, which is related to cancer and inflammation. Binding of SN-1 to TRAF6 and its effect on TRAF6 ubiquitination were examined in vitro, and the binding mode was calculated by computational methodology. Furthermore, ubiquitination of TRAF6 and downstream signaling was examined by cell-based experiments. The results show that SN-1 binds to TRAF6, inhibiting its auto-ubiquitination and downstream NF-κB signaling. Docking studies indicate that SN-1 binds directly to the first zinc finger of TRAF6. This binding disrupts the neighboring structure, that is, the RING finger domain, to suppress the ubiquitin ligase activity of TRAF6. Taken together, this study provides a platform for developing new small molecules that target zinc finger proteins.

TRIM25 in the Regulation of the Antiviral Innate Immunity.

TRIM25 is an E3 ubiquitin ligase enzyme that is involved in various cellular processes, including regulation of the innate immune response against viruses. TRIM25-mediated ubiquitination of the cytosolic pattern recognition receptor RIG-I is an essential step for initiation of the intracellular antiviral response and has been thoroughly documented. In recent years, however, additional roles of TRIM25 in early innate immunity are emerging, including negative regulation of RIG-I, activation of the melanoma differentiation-associated protein 5–mitochondrial antiviral signaling protein–TRAF6 antiviral axis and modulation of p53 levels and activity. In addition, the ability of TRIM25 to bind RNA may uncover new mechanisms by which this molecule regulates intracellular signaling and/or RNA virus replication.

Structural Characterization of the Trimerization of TRAF6 Protein Through Molecular Dynamics Simulations.

The tumour necrosis factor (TNF) receptor-associated factor (TRAF) family of proteins having E3 ligase activity are the key molecules involved in cellular immune response pathways. TRAF6 is a unique member of the TRAF superfamily differing from other members of the family, owing to its specific interactions with molecules outside the TNF receptor superfamily. The C-terminal domain of TRAF proteins contains the catalytic residues and are known to be involved in self-oligomerization forming a mushroom-shaped trimeric structure, which is the functional form of the protein. However, the monomeric crystal structure of TRAF6 C-terminal domain has been already determined, but the trimeric structure of the same is still not available. We here applied computational structural modelling and molecular dynamics simulations studies to get insights into the molecular interactions involved in determining the trimeric structure of the TRAF6 C-terminal domain. The non-availability of the trimeric structure of the TRAF6 C-terminal domain prevented the elucidation of the molecular mechanism of many different biological processes. Our results suggest that the trimer complex is transient in nature. The amino acid residues Lys340 and Glu345 in the coiled coil domain in the C-terminus of TRAF6 play a critical role in trimer structure formation. This structural modelling study may therefore be utilized to obtain the experimentally validated trimeric structure of this important protein.

Structure-function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune-suppressing activity from recognition.

Summary Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I‐2 carrying tomato (Solanum lycopersicum). Fol strains that evade I‐2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence.Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild‐type ΔspAvr2 (deleted signal‐peptide) or the ΔspAvr2 R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity‐related readouts, including flg22‐induced growth inhibition, ROS production and callose deposition.The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici‐repentis and to TRAF proteins. The I‐2 resistance‐breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface‐presented loop. Structure‐guided mutagenesis enabled uncoupling of virulence from I‐2‐mediated recognition.We conclude that I‐2‐mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope.

Regulation of RANKL-induced osteoclastogenesis by RING finger protein RNF114.

Normal bone remodeling is a continuous process orchestrated by bone-resorbing osteoclasts and bone-forming osteoblasts, which an imbalance in bone remodeling results in metabolic bone diseases. RANKL, a member of the TNF cytokine family, functions as a key stimulator for osteoclast differentiation and maturation. Here, we report that RNF114, previously identified as a psoriasis susceptibility gene, plays a regulatory role in the RANKL/RANK/TRAF6 signaling pathway that mediates osteoclastogenesis. Our results demonstrated that RNF114 expression was significantly down-regulated in mouse osteoclast precursor cells undergoing RANKL-induced osteoclast differentiation. RNF114 knockout did not affect development or viability of the subpopulation of bone marrow macrophages capable of differentiating into osteoclasts in culture. However, in the presence of RANKL, RNF114 knockout bone marrow macrophages exhibited enhanced cell proliferation and augmented osteoclast differentiation, as shown by an increased expression of mature osteoclast markers, increased osteoclastic TRAP activity and bone resorption. Conversely, ectopic expression of RNF114 inhibited CTSK expression, TRAP activity, and bone resorption in RANKL-treated pre-osteoclasts. RNF114 also suppressed RANKL-activated NFATc1 expression and NFAT-regulated promoter activity. RNF114 suppressed TRAF6-, but not TAK1/TAB2-mediated NF-κB activation downstream of RANKL/RANK. In particular, TRAF6 protein levels were down-regulated by RNF114, possibly via K48-mediated proteasome-dependent degradation. These data suggested that RNF114's inhibitory effect on RANKL-stimulated osteoclastogenesis was mediated by blocking RANK/TRAF6/NF-κB signal transduction. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:159-166, 2018.

Intervention of Dietary Dipeptide Gamma-l-Glutamyl-l-Valine (γ-EV) Ameliorates Inflammatory Response in a Mouse Model of LPS-Induced Sepsis.

Sepsis, the systemic inflammatory response syndrome (SIRS) with infection is one of the leading causes of death in critically ill patients in the developed world due to the lack of effective antisepsis treatments. This study examined the efficacy of dietary dipeptide gamma-l-glutamyl-l-valine (γ-EV), which was characterized previously as an anti-inflammatory peptide, in an LPS-induced mouse model of sepsis. BALB/c mice were administered γ-EV via oral gavage followed by an intraperitoneal injection of LPS to induce sepsis. The γ-EV exhibited antisepsis activity by reducing the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in plasma and small intestine. γ-EV also reduced the phosphorylation of the signaling proteins JNK and IκBα. We concluded that γ-EV could possess an antisepsis effect against bacterial infection in intestine. This study proposes a signaling mechanism whereby the calcium-sensing receptor (CaSR) allosterically activated by γ-EV stimulates the interaction of β-arrestin2 with the TIR(TLR/IL-1R) signaling proteins TRAF6, TAB1, and IκBα to suppress inflammatory signaling.

Abstract 2524: MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells

Abstract Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date. Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3’UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression. Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3’UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124. Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells. Citation Format: Anita K. Mehta, Kevin Hua, William Whipple, Mihn-Thuy Nguyen, Rushika M. Perera, Johanns Haybaeck, Joanne Weidhass, Jeffrey Settleman, Anurag Singh. MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2017-2524