TRAF3 Interacting Protein 2 Research Articles

TRAF3IP3 at the trans-Golgi network regulates NKT2 maturation via MEK/ERK signaling pathway

Abstract Thymic natural killer T (NKT)2 cells are a subset of invariant NKT cells with PLZFhiGATA3hiIL-4+. The differentiation of NKT2 cells is not fully understood. We report in the present study an important role of TRAF3-interacting protein 3 (TRAF3IP3) in the functional maturation and expansion of committed NKT2s in thymic medulla. Mice with T cell-specific deletion of TRAF3IP3 had decreased thymic NKT2 cells, decreased IL-4 producing peripheral iNKTs, and defects in response to α-galactosylceramide. Positive selection and high PLZF expression in CD24+CD44− and CCR7+CD44− immature iNKTs were not affected. Only CD44hiNK1.1− iNKTs in Traf3ip3−/− mice showed reduced expression of Egr2, PLZF, IL-17RB, decreased proliferation, and decreased IL-4 production upon stimulation. Such Egr2 and IL-4 expression were augmented by MEK1/ERK activation in iNKTs and TRAF3IP3 at the trans-Golgi network recruited MEK1 and facilitated ERK phosphorylation and nuclear translocation. These data demonstrate an important functional maturation process in NKT2 differentiation that is regulated by MEK/ERK signaling at the trans-Golgi network.

RECK suppresses interleukin‐17/TRAF3IP2‐mediated MMP‐13 activation and human aortic smooth muscle cell migration and proliferation

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular proliferative/occlusive disorders. The proinflammatory cytokine interleukin (IL)‐17 signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various transcription factors critical for inflammatory responses, including AP‐1 and NF‐κB. Here we investigated whether IL‐17A/TRAF3IP2 signaling promotes MMP‐13‐dependent human aortic smooth muscle cell (SMC) migration and proliferation, and whether RECK overexpression blunts its mitogenic and migratory potential. Indeed, IL‐17A treatment induced (i) AP‐1, NF‐κB, CREB and p38 MAPK activation, (ii) miR‐21 induction, (iii) miR‐27b and miR‐320 inhibition, (iv) MMP‐13 expression and activation, (v) RECK suppression, and (vi) SMC migration and proliferation, all in a TRAF3IP2‐dependent manner. RECK (REversion‐inducing‐Cysteine‐rich protein with Kazal motifs) is a membrane‐anchored MMP inhibitor, and forced expression of TRAF3IP2 suppressed its expression, but induced MMP‐13 expression and activation. Importantly, forced expression of RECK promoted IL‐17‐mediated MMP‐13/RECK physical association, reduced MMP13 activity and inhibited MMP‐13‐dependent SMC migration and proliferation. Further supporting its causative role in vascular proliferative diseases, addition of recombinant MMP‐13 stimulated SMC migration and proliferation, in part via ERK activation. These results indicate that increased MMP‐13 and decreased RECK contribute to IL‐17A/TRAF3IP2‐mediated SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases.Support or Funding InformationVA‐I01‐BX002255IK6BX004016‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRAF3IP2 mediates TWEAK/TWEAKR-induced pro-fibrotic responses in cultured cardiac fibroblasts and the heart

Persistent inflammation promotes development and progression of heart failure (HF). TWEAK (TNF-Related WEAK Inducer Of Apoptosis), a NF-κB- and/or AP-1-responsive proinflammatory cytokine that signals via TWEAK receptor (TWEAKR), is expressed at high levels in human and preclinical models of HF. Since the adapter molecule TRAF3IP2 (TRAF3 Interacting Protein 2) is an upstream regulator of various proinflammatory pathways, including those activated by NF-κB and AP-1, we hypothesized that targeting TRAF3IP2 inhibits TWEAK-induced proinflammatory and pro-fibrotic responses in vitro and in vivo. Consistent with the hypothesis, forced expression of TRAF3IP2 upregulated TWEAK and its receptor expression in cultured adult mouse cardiac fibroblasts (CF). Further, exogenous TWEAK upregulated TRAF3IP2 expression in a time- and dose-dependent manner, suggesting a positive-feedback regulation of TRAF3IP2 and TWEAK. TWEAK also promoted TRAF3IP2 nuclear translocation. Confirming its critical role in TWEAK signaling, silencing TRAF3IP2 inhibited TWEAK autoregulation, TWEAKR upregulation, p38 MAPK, NF-κB and AP-1 activation, inflammatory cytokine expression, MMP and TIMP1 activation, collagen expression and secretion, and importantly, proliferation and migration. Recapitulating these in vitro results, continuous infusion of TWEAK for 7 days increased systolic blood pressure (SBP), upregulated TRAF3IP2 expression, activated p38 MAPK, NF-κB and AP-1, induced the expression of multiple proinflammatory and pro-fibrotic mediators, and interstitial fibrosis in hearts of wild type mice. These proinflammatory and pro-fibrotic changes occurred in conjunction with myocardial hypertrophy and contractile dysfunction. Importantly, genetic ablation of TRAF3IP2 inhibited these TWEAK-induced adverse cardiac changes independent of increases in SBP, indicating that TRAF3IP2 plays a causal role, and thus a therapeutic target, in chronic inflammatory and fibro-proliferative diseases.

TRAF3IP2, a novel therapeutic target in glioblastoma multiforme.

Glioblastoma multiforme (glioblastoma) remains one of the deadliest cancers. Pro-inflammatory and pro-tumorigenic mediators present in tumor microenvironment (TME) facilitate communication between tumor cells and adjacent non-malignant cells, resulting in glioblastoma growth. Since a majority of these mediators are products of NF-κB- and/or AP-1-responsive genes, and as TRAF3 Interacting Protein 2 (TRAF3IP2) is an upstream regulator of both transcription factors, we hypothesized that targeting TRAF3IP2 blunts tumor growth by inhibiting NF-κB and pro-inflammatory/pro-tumorigenic mediators. Our in vitro data demonstrate that similar to primary glioblastoma tumor tissues, malignant glioblastoma cell lines (U87 and U118) express high levels of TRAF3IP2. Silencing TRAF3IP2 expression inhibits basal and inducible NF-κB activation, induction of pro-inflammatory mediators, clusters of genes involved in cell cycle progression and angiogenesis, and formation of spheroids. Additionally, silencing TRAF3IP2 significantly increases apoptosis. In vivo studies indicate TRAF3IP2-silenced U87 cells formed smaller tumors. Additionally, treating existing tumors formed by wild type U87 cells with lentiviral TRAF3IP2 shRNA markedly regresses their size. Analysis of residual tumors revealed reduced expression of pro-inflammatory/pro-tumorigenic/pro-angiogenic mediators and kinesins. In contrast, the expression of IL-10, an anti-inflammatory cytokine, was increased. Together, these novel data indicate that TRAF3IP2 is a master regulator of malignant signaling in glioblastoma, and its targeting modulates the TME and inhibits tumor growth by suppressing the expression of mediators involved in inflammation, angiogenesis, growth, and malignant transformation. Our data identify TRAF3IP2 as a potential therapeutic target in glioblastoma growth and dissemination.

TRAF3-interacting protein 3, a new oncotarget, promotes tumor growth in melanoma.

TRAF3-interacting protein 3 (TRAF3IP3) is expressed in the immune system and participates in cell maturation, tissue development, and immune response. In a previous study, we reported that TRAF3IP3 levels were substantially increased in the vasculature of breast cancer tissues, suggesting a proangiogenic role. In this study, we investigated TRAF3IP3 tumorigenic function. TRAF3IP3 protein was present in several cancer cell lines, with highest levels in melanoma. In addition, tumor microarray analysis on 23 primary melanoma and nine positive lymph nodes revealed that 70% of human primary melanoma and 66% of lymph node metastases were positive for TRAF3IP3. Importantly, TRAF3IP3 downregulation correlated with an 83% reduction of tumor growth in a subcutaneous xenograft mouse model (n=10, P=0.005). Immunohistochemistry analysis of the tumors revealed that TRAF3IP3-shRNA tumors had increased apoptosis (n=4, P<0.01) and reduced microvascular density (n=4, P<0.002). In addition, TRAF3IP3 downregulation in malignant endothelial cells reduced tube formation in a Matrigel tube formation assay. In melanoma cells, decreased levels of TRAF3IP3 were also associated with reduced viability (n=4, P=0.03) and proliferation (n=3, P=0.03), together with increased sensitivity to ultraviolet-induced apoptosis (n=4, P=0.0004). Furthermore, TRAF3IP3 downregulation correlated with increased amounts of interferon-γ. Interferon-γ inhibits tumor growth and angiogenesis, thus suggesting a new pathway for TRAF3IP3 in cancer. Collectively, the association of TRAF3IP3 with malignant properties of melanoma suggest a clinical potential for targeted therapy.

TRAF3IP2 mediates high glucose-induced endothelin-1 production as well as endothelin-1-induced inflammation in endothelial cells.

Hyperglycemia-induced production of endothelin (ET)-1 is a hallmark of endothelial dysfunction in diabetes. Although the detrimental vascular effects of increased ET-1 are well known, the molecular mechanisms regulating endothelial synthesis of ET-1 in the setting of diabetes remain largely unidentified. Here, we show that adapter molecule TRAF3 interacting protein 2 (TRAF3IP2) mediates high glucose-induced ET-1 production in endothelial cells and ET-1-mediated endothelial cell inflammation. Specifically, we found that high glucose upregulated TRAF3IP2 in human aortic endothelial cells, which subsequently led to activation of JNK and IKKβ. shRNA-mediated silencing of TRAF3IP2, JNK1, or IKKβ abrogated high-glucose-induced ET-converting enzyme 1 expression and ET-1 production. Likewise, overexpression of TRAF3IP2, in the absence of high glucose, led to activation of JNK and IKKβ as well as increased ET-1 production. Furthermore, ET-1 transcriptionally upregulated TRAF3IP2, and this upregulation was prevented by pharmacological inhibition of ET-1 receptor B using BQ-788, or inhibition of NADPH oxidase-derived reactive oxygen species using gp91ds-tat and GKT137831. Notably, we found that knockdown of TRAF3IP2 abolished ET-1-induced proinflammatory and adhesion molecule (IL-1β, TNF-α, monocyte chemoattractant protein 1, ICAM-1, VCAM-1, and E-selectin) expression and monocyte adhesion to endothelial cells. Finally, we report that TRAF3IP2 is upregulated and colocalized with CD31, an endothelial marker, in the aorta of diabetic mice. Collectively, findings from the present study identify endothelial TRAF3IP2 as a potential new therapeutic target to suppress ET-1 production and associated vascular complications in diabetes. NEW & NOTEWORTHY This study provides the first evidence that the adapter molecule TRAF3 interacting protein 2 mediates high glucose-induced production of endothelin-1 by endothelial cells as well as endothelin-1-mediated endothelial cell inflammation. The findings presented herein suggest that TRAF3 interacting protein 2 may be an important therapeutic target in diabetic vasculopathy characterized by excess endothelin-1 production.

Dipeptidyl Peptidase-4 Inhibition With Saxagliptin Ameliorates Angiotensin II-Induced Cardiac Diastolic Dysfunction in Male Mice.

Activation of the renin-angiotensin-aldosterone system is common in hypertension and obesity and contributes to cardiac diastolic dysfunction, a condition for which no treatment currently exists. In light of recent reports that antihyperglycemia incretin enhancing dipeptidyl peptidase (DPP)-4 inhibitors exert cardioprotective effects, we examined the hypothesis that DPP-4 inhibition with saxagliptin (Saxa) attenuates angiotensin II (Ang II)-induced cardiac diastolic dysfunction. Male C57BL/6J mice were infused with either Ang II (500 ng/kg/min) or vehicle for 3 weeks receiving either Saxa (10 mg/kg/d) or placebo during the final 2 weeks. Echocardiography revealed Ang II-induced diastolic dysfunction, evidenced by impaired septal wall motion and prolonged isovolumic relaxation, coincident with aortic stiffening. Ang II induced cardiac hypertrophy, coronary periarterial fibrosis, TRAF3-interacting protein 2 (TRAF3IP2)-dependent proinflammatory signaling [p-p65, p-c-Jun, interleukin (IL)-17, IL-18] associated with increased cardiac macrophage, but not T cell, gene expression. Flow cytometry revealed Ang II-induced increases of cardiac CD45+F4/80+CD11b+ and CD45+F4/80+CD11c+ macrophages and CD45+CD4+ lymphocytes. Treatment with Saxa reduced plasma DPP-4 activity and abrogated Ang II-induced cardiac diastolic dysfunction independent of aortic stiffening or blood pressure. Furthermore, Saxa attenuated Ang II-induced periarterial fibrosis and cardiac inflammation, but not hypertrophy or cardiac macrophage infiltration. Analysis of Saxa-induced changes in cardiac leukocytes revealed Saxa-dependent reduction of the Ang II-mediated increase of cardiac CD11c messenger RNA and increased cardiac CD8 gene expression and memory CD45+CD8+CD44+ lymphocytes. In summary, these results demonstrate that DPP-4 inhibition with Saxa prevents Ang II-induced cardiac diastolic dysfunction, fibrosis, and inflammation associated with unique shifts in CD11c-expressing leukocytes and CD8+ lymphocytes.

Targeting TRAF3IP2 by Genetic and Interventional Approaches Inhibits Ischemia/Reperfusion-induced Myocardial Injury and Adverse Remodeling

Re-establishing blood supply is the primary goal for reducing myocardial injury in subjects with ischemic heart disease. Paradoxically, reperfusion results in nitroxidative stress and a marked inflammatory response in the heart. TRAF3IP2 (TRAF3 Interacting Protein 2; previously known as CIKS or Act1) is an oxidative stress-responsive cytoplasmic adapter molecule that is an upstream regulator of both IκB kinase (IKK) and c-Jun N-terminal kinase (JNK), and an important mediator of autoimmune and inflammatory responses. Here we investigated the role of TRAF3IP2 in ischemia/reperfusion (I/R)-induced nitroxidative stress, inflammation, myocardial dysfunction, injury, and adverse remodeling. Our data show that I/R up-regulates TRAF3IP2 expression in the heart, and its gene deletion, in a conditional cardiomyocyte-specific manner, significantly attenuates I/R-induced nitroxidative stress, IKK/NF-κB and JNK/AP-1 activation, inflammatory cytokine, chemokine, and adhesion molecule expression, immune cell infiltration, myocardial injury, and contractile dysfunction. Furthermore, Traf3ip2 gene deletion blunts adverse remodeling 12 weeks post-I/R, as evidenced by reduced hypertrophy, fibrosis, and contractile dysfunction. Supporting the genetic approach, an interventional approach using ultrasound-targeted microbubble destruction-mediated delivery of phosphorothioated TRAF3IP2 antisense oligonucleotides into the LV in a clinically relevant time frame significantly inhibits TRAF3IP2 expression and myocardial injury in wild type mice post-I/R. Furthermore, ameliorating myocardial damage by targeting TRAF3IP2 appears to be more effective to inhibiting its downstream signaling intermediates NF-κB and JNK. Therefore, TRAF3IP2 could be a potential therapeutic target in ischemic heart disease.

Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPβ and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPβ, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction.

TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE−/− mice

Background and aimsAtherosclerosis is a major cause of heart attack and stroke. Inflammation plays a critical role in the development of atherosclerosis. Since the cytoplasmic adaptor molecule TRAF3IP2 (TRAF3-Interacting Protein 2) plays a causal role in various autoimmune and inflammatory diseases, we hypothesized that TRAF3IP2 mediates atherosclerotic plaque development. MethodsTRAF3IP2/ApoE double knockout (DKO) mice were generated by crossing TRAF3IP2−/− and ApoE−/− mice. ApoE−/− mice served as controls. Both DKO and control mice were fed a high-fat diet for 12 weeks. Plasma lipids were measured by ELISA, atherosclerosis by en face analysis of aorta and plaque cross-section measurements at the aortic valve region, plaque necrotic core area, collagen and smooth muscle cell (SMC) content by histomorphometry, and aortic gene expression by RT-qPCR. ResultsThe plasma lipoprotein profile was not altered by TRAF3IP2 gene deletion in ApoE−/− mice. While total aortic plaque area was decreased in DKO female, but not male mice, the plaque necrotic area was significantly decreased in DKO mice of both genders. Plaque collagen and SMC contents were increased significantly in both female and male DKO mice compared to respective controls. Aortic expression of proinflammatory cytokine (Tumor necrosis factor α, TNFα), chemokine (Chemokine (C-X-C motif) Ligand 1, CXCL1) and adhesion molecule (Vascular cell adhesion molecule 1, VCAM1; and Intercellular adhesion molecule 1, ICAM1) gene expression were decreased in both male and female DKO mice. In addition, the male DKO mice expressed markedly reduced levels of extracellular matrix (ECM)-related genes, including TIMP1 (Tissue inhibitor of metalloproteinase 1), RECK (Reversion-Inducing-Cysteine-Rich Protein with Kazal Motifs) and ADAM17 (A Disintegrin And Metalloproteinase 17). ConclusionsTRAF3IP2 plays a causal role in atherosclerotic plaque development and vulnerability, possibly by inducing the expression of multiple proinflammatory mediators. TRAF3IP2 could be a potential therapeutic target in atherosclerotic vascular diseases.

TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3 Interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2 mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Further, aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. However, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen IIIα1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) mRNA expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease.

T cell development involves TRAF3IP3-mediated ERK signaling in the Golgi.

Generation of T lymphocytes in the thymus is guided by signal transduction from the T cell receptor (TCR), but the underlying mechanism is incompletely understood. Here we have identified a Golgi-associated factor, TRAF3-interacting protein 3 (TRAF3IP3), as a crucial mediator of thymocyte development. TRAF3IP3 deficiency in mice attenuates the generation of mature thymocytes caused by impaired thymocyte-positive selection. TRAF3IP3 mediates TCR-stimulated activation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) and its upstream kinase mitogen/extracellular signal-regulated kinase (MEK). Interestingly, TRAF3IP3 exerts this signaling function through recruiting MEK to the Golgi and, thereby, facilitating the interaction of MEK with its activator BRAF. Transgenic expression of a constitutively active MEK rescues the T cell development block in Traf3ip3 knockout mice. These findings establish TRAF3IP3 as a novel regulator of T cell development and suggest a Golgi-specific ERK signaling mechanism that regulates thymocyte development.

Abstract 19428: TRAF3IP2 Mediates Aldosterone-induced Cardiac Hypertrophy and Fibrosis

Background: Aldosterone (Aldo) is a key regulator of blood pressure and electrolyte balance. Aldo/salt has been shown to induce oxidative stress, inflammation, cardiac hypertrophy and fibrosis. TRAF3 interacting protein 2 (TRAF3IP2) is a cytoplasmic redox-sensitive adaptor molecule and an upstream regulator of NF-κB and AP-1. It plays a critical role in the regulation of various proinflammatory cytokines that exert prohypertrophic and profibrotic effects. Therefore, we hypothesized that TRAF3IP2 is a critical mediator of Aldo/salt-induced cardiac hypertrophy and fibrosis. Methods: TRAF3IP2-null and wild type (WT) mice (male, 12-13 weeks) were infused continuously with Aldo for 4 weeks via mini-osmotic pumps (0.2 mg/Kg/day; n = 6-8). Mice were fed 1% NaCl in drinking water. Salt alone served as control. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography, myocardial function by echocardiography, cardiac hypertrophy by heart weight to body weight ratios, cardiac fibrosis by trichrome Gomori, and gene and protein expression by RT-qPCR, immunoblotting and ELISA. Results: Aldo/salt elevated SBP to a similar extent in both TRAF3IP2-null and WT mice. Aldo/salt upregulated TRAF3IP2 expression in left ventricular homogenates from WT mice, and TRAF3IP2 gene deletion (vs. WT) blunted cardiac hypertrophy by 54% (p &lt; 0.05), suppressed ANP expression by 3.5 fold (p &lt; 0.05), prevented concentric LV remodeling (decreased LVPWs; 93%; p &lt; 0.01, LVPWd; 95%; p &lt; 0.05, LVAWs; 29.8%; p &lt; 0.05 and LVAWd; 26.2%, and increased LVIDs; 19.2%; p &lt; 0.01 and LVIDd; 8.53%) and markedly reduced both interstitial and perivascular fibrosis. Further, TRAF3IP2 gene deletion inhibited Aldo/salt-induced p65 and c-Jun activation, and expression of inflammatory cytokine (IL-18 and IL-6), matrix metalloproteinase (MMP2) and extracellular matrix proteins (collagen Iα1 and IIIα1). TRAF3IP2 gene deletion also blunted mRNA and protein expression of lysyl oxidase, an enzyme involved in collagen crosslinking. Conclusions: TRAF3IP2 gene deletion blunts Aldo/salt-induced cardiac hypertrophy and fibrosis by inhibiting proinflammatory, prohypertrophic, and profibrotic effects, signifying its potential as a therapeutic target in hypertrophic heart diseases.

SUMOylation is required for optimal TRAF3 signaling capacity.

TNF receptor–associated factors (TRAFs) are multifunctional adaptor proteins involved in temporal and spatial coordination of signals necessary for normal immune function. Here, we report that TRAF3, a TRAF family member with a key role in Toll-like and TNF family receptor signaling and suppressor of lymphomagenesis, is post-translationally modified by the small ubiquitin-related modifier (SUMO). Through yeast two-hybrid and co-immunoprecipitation assays we have identified Ubc9, the SUMO conjugating enzyme, as a novel TRAF3-interacting protein. We show that Ubc9-dependent SUMOylation of TRAF3 modulates optimal association with the CD40 receptor, thereby influencing TRAF3 degradation and non-canonical NF-κB activation upon CD40 triggering. Collectively, our findings describe a novel post-translational modification of a TRAF family member and reveal a link between SUMOylation and TRAF-mediated signal transduction.

Patterns of evolutionary selection pressure in the immune signaling protein TRAF3IP2 in mammals

TRAF3 interacting protein 2 (TRAF3IP2) is important for immune responses to pathogens, inflammatory signals and autoimmunity in mammals. In the present study, we collected 19 mammalian TRAF3IP2 sequences and investigated the various types of selection pressure acting on them. Maximum likelihood estimations of nonsynonymous (dN) to synonymous (dS) substitution (dN/dS) ratios for the aligned coding sequences indicated that, as a whole, TRAF3IP2 has been subject to purifying selection. However, the N-terminus of the protein has been subject to higher selection pressure than the C-terminal domain. While eight amino acid residues within the N-terminus appear to have evolved under positive selection, no evidence for such selection was found in the C-terminus. The positively selected residues, which fall outside the currently known functional sites within TRAF3IP2, may have novel functions. The different selection pressures acting on the N- and C-terminal regions are consistent with their protein structures: the C-terminal structure is an ordered structure, whereas the N-terminus is disordered. Taken together with the results of previous studies, it is plausible that positive selection on the N-terminus of TRAF3IP2 may have occurred by competitive coevolution between mammalian hosts and viruses.

Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. These results demonstrate that the promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-xpressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo.

Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling

Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Because elevated AOPPs serve as independent risk factors for ischemic heart disease, and cardiomyocyte death is a hallmark of ischemic heart disease, we hypothesized that AOPPs will induce cardiomyocyte death. AOPP-modified mouse serum albumin (AOPP-MSA) induced significant death of neonatal mouse cardiomyocytes that was attenuated by knockdown of the receptor for advanced glycation end products, but not CD36. Notably, TRAF3-interacting protein 2 (TRAF3IP2; also known as CIKS or Act1) knockdown blunted AOPP-induced apoptosis. AOPP-MSA stimulated Nox2/Rac1-dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2-dependent JNK activation. The superoxide anion generating xanthine/xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPP-MSA induced mitochondrial Bax translocation and release of cytochrome c into cytoplasm. Moreover, AOPP-MSA suppressed antiapoptotic Bcl-2 and Bcl-xL expression. These effects were reversed by TRAF3IP2 knockdown or forced expression of mutant JNK. Similar to its effects in neonatal cardiomyocytes, AOPP-MSA induced adult cardiomyocyte death in part via TRAF3IP2. These results demonstrate for the first time that AOPPs induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK activation in vitro and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 may represent a potential therapeutic target in ischemic heart disease.