Tribbles Homolog Research Articles

Regulation and Functional Studies of Tribbles homolog 2 (TRIB2) in Ovarian Granulosa Cells

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2 mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate TRIB2 mRNA and protein regulation, to identify its binding partners, and study its function in GC of bovine dominant follicles. Granulosa cells were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the estrous), ovulatory follicles (OF) 24 h following injection of an ovulatory dose of hCG, and corpus luteum (CL) at day 5 of the estrous cycle. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF (P < 0.0001). Temporal expression of TRIB2 mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 h after hCG injection. There was a 20‐ and 166‐fold reduction of TRIB2 steady‐state mRNA levels in follicular walls, respectively at 6 and 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by LH/hCG at the protein level. Yeast two‐hybrid screening of a DF‐cDNA library as well as functional studies using the CRISPR‐Cas9 approach are currently being used to identify TRIB2 binding partners and its function in GC. These results support a physiologically relevant role of TRIB2 in follicular development and GC function.Support or Funding InformationResearch supported by internal funds from Université de Montréal to KN and NSERC of Canada grant # 104199 to JGL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High glucose contributes to the proliferation and migration of non-small cell lung cancer cells via GAS5-TRIB3 axis.

Despite the growing number of studies exhibiting an association of diabetes mellitus (DM) and lung cancer progression, the concrete mechanism of DM aggravating lung cancer has not been elucidated. The present study was to investigate whether and how high glucose (HG) contributes to the proliferation and migration of non-small-cell lung cancer (NSCLC) cells in vitro. In the present study, we confirmed that HG promoted the proliferation and migration of NSCLC cells, and also induced an anti-apoptotic effect on NSCLC cells. Moreover, HG inhibited the expression of growth arrest-specific 5 (GAS5) in NSCLC cells but elevated the protein level of tribbles homolog 3 (TRIB3). GAS5 overexpression promoted the degradation of TRIB3 protein by ubiquitination and inhibited the HG-induced proliferation, anti-apoptosis, and migration of NSCLC cells. Importantly, TRIB3 overexpression reversed the effects of GAS5 on the HG-treated NSCLC cells. Taken together, down-regulated GAS5 by HG significantly enhanced the proliferation, anti-apoptosis, and migration in NSCLC cells through TRIB3, thus promoting the carcinogenesis of NSCLC.

Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

Bone formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the bone marrow. Microgravity in spaceflight is known to reduce bone formation. In this study, we used a real microgravity environment of the SJ-10 Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-10 satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of 10 genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding protein β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of bone morphogenetic protein-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and protein kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased protein expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

TRIB2 knockdown as a regulator of chemotherapy resistance and proliferation via the ERK/STAT3 signaling pathway in human chronic myelogenous leukemia K562/ADM cells.

Acquired resistance to chemotherapy plays a critical role in human drug treatment failure in many tumor types. Multidrug resistance (MDR) to Adriamycin (ADM) also limits the efficacy of therapy in human chronic myelogenous leukemia (CML). The overexpression of drug efflux transporters is one mechanism uderlying MDR. In particular, the consistent activation of MDR1 and MDR‑associated protein 1 (MRP1) is involved in drug resistance. In the present study, ADM‑resistant human CML K562/ADM cells were stably transfected with a Tribbles homolog 2 (TRIB2)‑targeted vector. A CCK‑8 assay showed that the half maximal inhibitory concentration (IC50) of ADM and the cell proliferation were lower in the transfected cells compared with that in the parental K562/ADM cells. The mRNA and protein expression levels of MDR1 and MRP1 were determined by reverse transcription‑polymerase chain reaction (RT‑PCR), RT‑quantitative PCR and western blot analysis. The results showed that the expression of MDR1 and MRP1 was significantly reduced in K562/ADM cells transfected with pGPU6/GFP/Neo‑TRIB2. Due to the downregulation of MDR1 and MRP1, the intracellular accumulation of ADM was increased in the transfected cells compared with that in the parental K562/ADM cells. Therefore, the sensitivity of the K562/ADM cells to ADM was enhanced and proliferation was inhibited. Our research revealed that protein expression of the ERK signaling pathway was inhibited by downregulating TRIB2, indicating that the ERK pathway was involved in cell drug resistance and proliferation. Furthermore, we used the ERK‑specific blocker U0126 to demonstrate this phenomenon. In summary, our research suggested that knockdown of TRIB2 could slow cell growth and reverse resistance, implying that TRIB2 is a potential therapy target for resistant human CML.

TRIB3 limits FGF21 induction during in vitro and in vivo nutrient deficiencies by inhibiting C/EBP–ATF response elements in the Fgf21 promoter

Mammals must be able to endure periods of limited food availability, and the liver plays a central role in the adaptation to nutritional stresses. TRIB3 (Tribbles homolog 3) is a cellular stress-inducible gene with a liver-centric expression pattern and it has been implicated in stress response regulation and metabolic control. In the current article, we study the involvement of TRIB3 in responses to nutrient deficiencies, including fasting for up to 48 h in mice. We show that hepatic expression of Trib3 is increased after 48 h of fasting and mice with a targeted deletion of the Trib3 gene present elevated hepatic triglyceride content and liver weight at 48 h, along with an upregulation of lipid utilization genes in the liver. Further, hepatic and serum levels of the metabolic stress hormone FGF21 are considerably increased in 48-h-fasted Trib3 knockout mice compared to wild type. Trib3 deficiency also leads to elevated FGF21 levels in the mouse liver during essential amino acid deficiency and in cultured mouse embryonic fibroblasts during glucose starvation. Reporter assays reveal that TRIB3 regulates FGF21 by inhibiting ATF4-mediated, C/EBP–ATF site-dependent activation of Fgf21 transcription. Based on chromatin immunoprecipitation from mouse liver, the binding of TRIB3 and ATF4, a transcription factor known to physically interact with TRIB3, is significantly increased at the Fgf21 promoter following 48 h of fasting. Thus, under nutrient-limiting conditions that stimulate ATF4 activity, TRIB3 is implicated in the regulation of metabolic adaptation by restraining the transcription of Fgf21.

Association of tribbles homologue 1 gene expression in human umbilical vein endothelial cells with duration of intrauterine exposure to hyperglycaemia.

Maternal gestational diabetes mellitus (GDM) is considered to be an important factor that epigenetically predisposes offspring to metabolic and cardiovascular diseases. However, the mechanisms of how intrauterine hyperglycaemia affects offspring have not been thoroughly studied. The mammalian tribbles homologue 1 (TRIB1) gene is associated with plasma lipid concentrations and coronary artery disease (CAD). Our aim was to study the effect of GDM and its treatment terms on the level of TRIB1 gene expression in human umbilical vein endothelial cells (HUVECs) of newborns from women with and without GDM. The study included 50 women with GDM and 25 women without GDM (control group). Women with GDM were divided into three groups according to their gestational age when the treatment of GDM started: 24-28 weeks (GDM1, N=16), 29-32 weeks (GDM2, N=25) and >34 weeks (GDM3, N=9). The levels of TRIB1 gene expression in GDM3, GDM2, GDM1 and control groups were 2.8±1.1, 4.2±2.4, 6.0±3.4 and 8.1±6.1, respectively (p=0.001). After comparison in pairs the difference was significant for the following pairs: GDM2-control (p=0.004), GDM3-control (p=0.002), GDM1-GDM3 (p=0.012). Notably, if treatment had been started before the 28th week of gestation, the difference in TRIB1 gene expression in HUVECs was not significant (p=0.320 for comparison between GDM1 and control groups). Our findings support the hypothesis that TRIB1 gene expression in HUVECs depends on the duration of intrauterine exposure to hyperglycaemia.

Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway

The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacAs1m1-induced cell death and apoptosis, and apoptosis inhibitor Z-VAD had no significant effect on autophagy induced by VacAs1m1. Thus, these results suggested that VacA causes autophagic cell death via ER stress in gastric epithelial cells.

TRB3 reverses chemotherapy resistance and mediates crosstalk between endoplasmic reticulum stress and AKT signaling pathways in MHCC97H human hepatocellular carcinoma cells.

Tribbles homolog 3 (TRB3), a type of pseudokinase that contains a consensus serine/threonine kinase catalytic core structure, is upregulated in hepatocellular carcinoma. However, the effect of TRB3 expression in hepatocellular carcinoma and the molecular mechanisms underlying TRB3-mediated effects on tumorigenesis in hepatocellular carcinoma have not been fully elucidated. The present study focused on the effect of TRB3 expression in MHCC97H hepatocellular carcinoma cells and investigated the underlying molecular mechanisms in MHCC97H cells. In the present study, it was revealed that TRB3 was significantly overexpressed in the MHCC97H hepatocellular carcinoma cell compared with L-02 normal hepatic cells. Under endoplasmic reticulum (ER) stress induced by thapsigargin and tunicamycin, the levels of TRB3, CCAAT/enhancer binding protein homologous protein (CHOP), protein kinase B (AKT) and phosphorylated (p)AKT expression were upregulated. Furthermore, when the expression of TRB3 was silenced by short hairpin (sh)RNA, the survival of MHCC97H hepatocellular carcinoma cells was increased. Notably, following transduction with lentiviral containing TRB3-shRNA, cell survival also increased after treatment with chemotherapy drug cisplatin. The present study demonstrated that knockdown of CHOP by shRNA was able to reduce TRB3 expression, and the knockdown of TRB3 markedly increased the level of pAKT. TRB3 was overexpressed in MHCC97H hepatocellular carcinoma cells, particularly under endoplasmic reticulum stress. Knockdown of TRB3 was able to increase cell survival. Therefore, TRB3 expression may induce apoptosis and reverse resistance to chemotherapy in MHCC97H hepatic carcinoma cells. The present study suggests that TRB3 is a key molecule that mediates the crosstalk between ER stress and AKT signal pathways. Furthermore, the present study may provide further insight into the cancer biology of hepatocellular carcinoma and the development of anticancer drugs targeting the ER stress and AKT signaling pathways.

Transcriptomic Profiling of MDA-MB-231 Cells Exposed to Boswellia Serrata and 3-O-Acetyl-B-Boswellic Acid; ER/UPR Mediated Programmed Cell Death.

Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors (estrogen, progesterone and human epidermal growth factor receptor-2) and a relatively poor prognosis due to inefficacy of hormone receptor-based chemotherapies. It is imperative that we continue to explore natural products with potential to impede growth and metastasis of TNBC. In this study, we screened over 1,000 natural products for capacity to induce cell death in TNBC (MDA-MB -231) cells. Frankincense (Boswellia serrata extract (BSE)) and 3-O-Acetyl-β-boswellic acid (3-OAβBA) were relatively potent, findings that corroborate the body of existing literature. The effects of BSE and 3-OAβBA on genetic parameters in MDA-MB-231 cells were evaluated by examining whole-transcriptomic influence on mRNAs, long intergenic non-coding RNA transcripts (lincRNA) and non-coding miRNAs. Bio-statistical analysis demarcates the primary effect of both BSE/3-OAβBA on the up-regulation of PERK (protein kinase RNA-like endoplasmic reticulum kinase)- endoplasmic reticulum (ER)/unfolded protein response (UPR) pathways that are closely tied to activated programmed cell death (APCD). Global profiling confirms concomitant effects of BSE/3-OAβBA on upwardly expressed ER/URP APCD key components PERK (EIF2AK3), XBP1, C/EBP homologous protein transcription factor (CHOP), ATF3 and DDIT3,4/DNA-damage-inducible transcript 3,4 (GADD34). Further, BSE and/or 3-OAβBA significantly down-regulated oncogenes (OG) which, heretofore, lack functional pathway mapping, but are capable of driving epithelial-mesenchymal transition (EMT), cell survival, proliferation, metastasis and drug resistance. Among these are cell migration-inducing protein hyaluronan binding (CEMIP) [-7.22]; transglutaminase 2 [-4.96], SRY box 9 (SOX9) [-4.09], inhibitor of DNA binding 1, dominant negative helix-loop-helix protein (ID1) [-6.56]; and endothelin 1 (EDN1, [-5.06]). Likewise, in the opposite manner, BSE and/or 3-OAβBA induced the robust overexpression of tumor suppressor genes (TSGs), including: glutathione-depleting ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) [+21.67]; the mTOR inhibitors - sestrin 2 (SESN2) [+16.4] Tribbles homolog 3 (TRIB3) [+6.2], homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERPUD1) [+12.01]; and cystathionine gamma-lyase (CTH) [+11.12]. The anti-cancer effects of the historically used frankincense sap (BSE) appear to involve major impact on the ER/UPR response, concomitant to effecting multiple targets counter to the growth, proliferation and metastasis of TNBC cancer cells. The microarray data are available at Expression Omnibus GEO Series accession number GSE102891.

Trib2 regulates the pluripotency of embryonic stem cells and enhances reprogramming efficiency

Embryonic stem (ES) cells are pluripotent cells characterized by self-renewability and differentiation potential. Induced pluripotent stem (iPS) cells are ES cell-equivalent cells derived from somatic cells by the introduction of core reprogramming factors. ES and iPS cells are important sources for understanding basic biology and for generating therapeutic cells for clinical applications. Tribbles homolog 2 (Trib2) functions as a scaffold in signaling pathways. However, the relevance of Trib2 to the pluripotency of ES and iPS cells is unknown. In the present study, we elucidated the importance of Trib2 in maintaining pluripotency in mouse ES cells and in generating iPS cells from somatic cells through the reprogramming process. Trib2 expression decreased as ES cells differentiated, and Trib2 knockdown in ES cells changed their colony morphology while reducing the activity of alkaline phosphatase and the expression of the pluripotency marker genes Oct4, Sox2, Nanog and Klf4. Trib2 directly interacted with Oct4 and elevated Oct4 promoter activity. During the generation of iPS cells, Trib2 knockdown decreased the reprogramming efficiency of mouse embryonic fibroblasts, whereas Trib2 overexpression significantly increased their reprogramming efficiency. In summary, our results suggest that Trib2 is important for maintaining self-renewal in ES cells and for pluripotency induction during the reprogramming process.

Deletion of protein tyrosine phosphatase 1B obliterates endoplasmic reticulum stress-induced myocardial dysfunction through regulation of autophagy

Endoplasmic reticulum (ER) stress has been demonstrated to prompt various cardiovascular risks although the underlying mechanism remains elusive. Protein tyrosine phosphatase-1B (PTP1B) serves as an essential negative regulator for insulin signaling. This study examined the role of PTP1B in ER stress-induced myocardial anomalies and underlying mechanism involved with a focus on autophagy. WT and PTP1B knockout mice were subjected to the ER stress inducer tunicamycin (1mg/kg). Cardiac function was evaluated with echocardiography and an Ion-Optix MyoCam system. Western blot analysis was used to monitor the levels of ER stress, autophagy and insulin signaling including insulin receptor substrate (IRS), tribbles homolog 3 (TRIB3), Atg5/7, p62 and LC3-II. Our results showed that ER stress resulted in compromised echocardiographic and cardiomyocyte contractile function, intracellular Ca2+ mishandling, ER stress, O2− production, apoptosis, the effects of which (with the exception of ER stress) were significantly attenuated or negated by PTP1B ablation. Levels of serine phosphorylation of IRS-1, TRIB3, Atg5/7, LC3B and the autophagy adaptor p62 were significantly upregulated while IRS-1 tyrosine phosphorylation was reduced by tunicamycin, the effect of which were obliterated by PTP1B ablation. In vitro study revealed that the autophagy inducer rapamycin and TRIB3 overexpression cancelled PTP1B ablation-offered beneficial effects on cardiomyocyte function or O2− production in murine cardiomyocytes or H9C2 myoblasts. Antioxidant or gene silencing of TRIB3 mimicked PTP1B ablation-induced protective effects. These findings collectively suggested that PTP1B ablation protects against ER stress-induced cardiac anomalies through regulation of autophagy.

TRIB3 inhibits proliferation and promotes osteogenesis in hBMSCs by regulating the ERK1/2 signaling pathway

Osteogenic differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) is regulated by various factors, including bone morphogenetic proteins (BMPs), Notch, growth hormones and mitogen-activated protein kinases (MAPKs). Tribbles homolog 3 (TRIB3), a pseudokinase, plays an important role in cancer cells and adipocytes. However, TRIB3 function in osteogenic differentiation is unknown, although it is involved in regulating signaling pathways associated with osteogenic differentiation. Here, we found that TRIB3 was highly expressed during osteogenic differentiation in hBMSCs. Inhibition of focal adhesion kinase (FAK) or phosphatidylinositol 3-kinase (PI3K) resulted in a significant decrease in TRIB3 expression, and expression of TRIB3 was restored by increasing insulin-like growth factor-1 (IGF-1) via activating phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling. TRIB3 knock-down enhanced proliferation and decreased osteogenic differentiation at the middle stage of differentiation, and these effects were reversed by inhibiting the activation of extracellular signal-regulated kinase (ERK)-1/2. In conclusion, TRIB3 plays an important role in proliferation and osteogenic differentiation by regulating ERK1/2 activity at the middle stage of differentiation, and expression of TRIB3 is regulated by FAK in a PI3K/AKT-dependent manner.

Small molecule-mediated tribbles homolog 3 promotes bone formation induced by bone morphogenetic protein-2

Although bone morphogenetic protein-2 (BMP2) has demonstrated extraordinary potential in bone formation, its clinical applications require supraphysiological milligram-level doses that increase postoperative inflammation and inappropriate adipogenesis, resulting in well-documented life-threatening cervical swelling and cyst-like bone formation. Recent promising alternative biomolecular strategies are toward promoting pro-osteogenic activity of BMP2 while simultaneously suppressing its adverse effects. Here, we demonstrated that small molecular phenamil synergized osteogenesis and bone formation with BMP2 in a rat critical size mandibular defect model. Moreover, we successfully elicited the BMP2 adverse outcomes (i.e. adipogenesis and inflammation) in the mandibular defect by applying high dose BMP2. Phenamil treatment significantly improves the quality of newly formed bone by inhibiting BMP2 induced fatty cyst-like structure and inflammatory soft-tissue swelling. The observed positive phenamil effects were associated with upregulation of tribbles homolog 3 (Trib3) that suppressed adipogenic differentiation and inflammatory responses by negatively regulating PPARγ and NF-κB transcriptional activities. Thus, use of BMP2 along with phenamil stimulation or Trib3 augmentation may be a promising strategy to improve clinical efficacy and safety of current BMP therapeutics.

Downregulation of TRB3 protects neurons against apoptosis induced by global cerebral ischemia and reperfusion injury in rats

Global cerebral ischemia and reperfusion injury (GCI/R) can lead to neuronal apoptosis and contributes to permanent neurological sequelae. However, the underlying mechanism is largely unknown. Therefore, the present study aimed to assess the effects of GCI/R on the tribbles homolog 3 (TRB3) and to explore the role of TRB3 in GCI/R. The GCI/R model was developed in Sprague–Dawley male rats by four-vessel occlusion. Subsequently, the expressions of TRB3, endoplasmic reticulum stress markers, and apoptosis-associated proteins were examined by western blot at 1h, 6h, 12h, 24h, and 72h after GCI/R. TRB3 short-hairpin RNA (shRNA) lentivirus was constructed and used to investigate the role of TRB3 in GCI/R-induced neuronal apoptosis. GCI/R increased the level of TRB3, endoplasmic reticulum stress markers, and pro-apoptotic proteins. The level of protein kinase B (Akt) phosphorylation was reduced during GCI/R. Administration of TRB3 shRNA lentivirus attenuated GCI/R-induced up-regulation of TRB3, endoplasmic reticulum stress, and neuronal apoptosis. Furthermore, TRB3 shRNA lentivirus reversed the reduced level of Akt phosphorylation induced by GCI/R. These data implied that TRB3 participated in the GCI/R-induced neuronal apoptosis. Knocking down TRB3 attenuated endoplasmic reticulum stress, enhanced Akt phosphorylation, and protected neurons from apoptosis in response to GCI/R. These results demonstrated that the downregulation of TRB3 may be a promising approach for treating GCI/R.

Severe hypertriglycerideamia related to a novel mutation in TRIB1 and a known variant in APOA5

Aim: To report a case with severe elevation of triglyceride (TG) levels who was found to have a novel mutation in tribbles homolog 1 (TRIB1) and an APOA5 variant.

Evaluation of Plasma TRB3 and Sestrin 2 Levels in Obese and Normal-Weight Children.

Obesity in childhood and adolescence is associated with metabolic syndrome and cardiovascular diseases. TRB3 (Tribbles homolog 3) and sestrin 2 are two newly found proteins that have been identified to play an important role in obesity and its complications. The purpose of this study was to evaluate concentrations of TRB3 and sestrin 2 in plasma of obese and normal-weight children and adolescents, and their association with metabolic and anthropometric parameters. Plasma levels of TRB3, sestrin 2, insulin, fasting plasma glucose (FPG), and lipid profile were evaluated in 70 children and adolescents (34 obese and 36 controls). Insulin resistance was calculated using a homeostasis model assessment of insulin resistance. Metabolic syndrome was defined according to IDF criteria. Plasma TRB3 levels of the obese subjects were significantly higher than that of normal weight subjects. TRB3 levels were positively correlated with BMI, BMI z-score, waist circumference, and FPG. The concentration of sestrin 2 was significantly lower in obese subjects compared to normal-weight subjects. A statistically significant positive correlation was observed between plasma concentrations of sestrin 2 and high-density lipoprotein cholesterol. Neither TRB3 nor sestrin 2 were correlated with insulin resistance and metabolic syndrome. Both TRB3 and sestrin 2 may contribute to the development of obesity and its complications and can be considered interesting therapeutic target for the treatment of obesity.

Tribbles homolog 1 enhances cholesterol efflux from oxidized low-density lipoprotein-loaded THP-1 macrophages.

Macrophage foam cell formation plays a pivotal role in the pathogenesis of atherosclerosis. The protein Tribbles homolog 1 (Trib1), a member of the Tribbles protein family, functions as an adaptor or scaffold protein. Recent studies have indicated its implication in lipoprotein metabolism. In the present study, the role of Trib1 in macrophage foam cell formation was investigated. Oil red O staining was used to analyze intracellular lipid deposition, while the effects of Trib1 overexpression on cholesterol efflux were also examined. Furthermore, quantitative polymerase chain reaction and western blot analysis were performed to measure the expression levels of genes involved in cholesterol efflux. The results revealed that overexpression of Trib1 inhibited lipid accumulation in oxidized low density lipoprotein-treated THP-1 macrophages and facilitated macrophage cholesterol efflux to apolipoprotein A-I. Overexpression of Trib1 also upregulated the expression levels of ATP-binding cassette A1 (ABCA1), ABCG1, liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ (PPARγ). In addition, silencing of LXRα or PPARγ via small interfering RNA transfection significantly reversed the Trib1-induced cholesterol efflux. In conclusion, Trib1 inhibits macrophage foam cell formation and enhances cholesterol efflux, which is associated with regulation of the PPARγ, LXRα, ABCA1 and ABCG1 expression levels.

Mammalian Pseudokinase TRIB3 in Normal Physiology and Disease: Charting the Progress in Old and New Avenues.

Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cell death-inducing cellular stress. The TRIB3 protein is a pseudokinase, a protein kinase-like scaffold with impaired catalytic activity. However, research has revealed it to be prolific at forming protein- protein interactions. By binding to and regulating the activity of several key proteins, including the protein kinase Akt and transcription factors ATF4, CHOP and NF-κB, TRIB3 is at a junction of several signaling pathways. This review begins by providing insights into the characteristic protein structure and gene expression regulation mechanisms of TRIB3. Further, the diverse reported molecular roles of TRIB3 as a regulator of cell death, stress responses, inflammation, cell differentiation, protein degradation and other processes are discussed. Special attention is devoted to the involvement of TRIB3 in the pathogenesis of cancer and type 2 diabetes, two fields where TRIB3 has generated particular interest, as well as considerable debate, from a biomedical standpoint. Throughout, emphasis is placed on results obtained from animal models with altered TRIB3 expression (Trib3 knockout or overexpression mice), in order to provide insight into the contributions of TRIB3 to physiology and disease at the organism level.

TRB3 mediates advanced glycation end product-induced apoptosis of pancreatic β-cells through the protein kinase C β pathway.

Advanced glycation end products (AGEs), which accumulate in the body during the development of diabetes, may be one of the factors leading to pancreatic β-cell failure and reduced β-cell mass. However, the mechanisms responsible for AGE-induced apoptosis remain unclear. This study identified the role and mechanisms of action of tribbles homolog 3 (TRB3) in AGE-induced β-cell oxidative damage and apoptosis. Rat insulinoma cells (INS-1) were treated with 200 µg/ml AGEs for 48 h, and cell apoptosis was then detected by TUNEL staining and flow cytometry. The level of intracellular reactive oxygen species (ROS) was measured by a fluorescence assay. The expression levels of receptor of AGEs (RAGE), TRB3, protein kinase C β2 (PKCβ2) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) were evaluated by RT-qPCR and western blot analysis. siRNA was used to knockdown TRB3 expression through lipofection, followed by an analysis of the effects of TRB3 on PKCβ2 and NOX4. Furthermore, the PKCβ2-specific inhibitor, LY333531, was used to analyze the effects of PKCβ2 on ROS levels and apoptosis. We found that AGEs induced the apoptosis of INS-1 cells and upregulated RAGE and TRB3 expression. AGEs also increased ROS levels in β-cells. Following the knockdown of TRB3, the AGE-induced apoptosis and intracellular ROS levels were significantly decreased, suggesting that TRB3 mediated AGE-induced apoptosis. Further experiments demonstrated that the knockdown of TRB3 decreased the PKCβ2 and NOX4 expression levels. When TRB3 was knocked down, the cells expressed decreased levels of PKCβ2 and NOX4. The PKCβ2-specific inhibitor, LY333531, also reduced AGE-induced apoptosis and intracellular ROS levels. Taken together, our data suggest that TRB3 mediates AGE-induced oxidative injury in β-cells through the PKCβ2 pathway.

TRB3 gene silencing activates AMPK in adipose tissue with beneficial metabolic effects in obese and diabetic rats

Our previous study had suggested Tribbles homolog 3 (TRB3) might be involved in metabolic syndrome via adipose tissue. Given prior studies, we sought to determine whether TRB3 plays a major role in adipocytes and adipose tissue with beneficial metabolic effects in obese and diabetic rats. Fully differentiated 3T3-L1 adipocytes were incubated to induce insulin resistant adipocytes. Forty male Sprague–Dawley rats were all fed high-fat (HF) diet. Type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin (STZ). Compared with control group, in insulin resistant adipocytes, protein levels of insulin receptor substrate-1(IRS-1), glucose transporter 4(GLUT4) and phosphorylated-AMP-activated protein kinase (p-AMPK)were reduced, TRB3 protein level and triglyceride level were significantly increased, glucose uptake was markedly decreased. TRB3 silencing alleviated adipocytes insulin resistance. With TRB3 gene silencing, protein levels of IRS-1, GLUT4 and p-AMPK were significantly increased in adipocytes. TRB3 gene silencing decreased blood glucose, ameliorated insulin sensitivity and adipose tissue remodeling in diabetic rats. TRB3 silencing decreased triglyceride, increased glycogen simultaneously in diabetic epididymal and brown adipose tissues (BAT). Consistently, p-AMPK levels were increased in diabetic epididymal adipose tissue, and BAT after TRB3-siRNA treatment. TRB3silencing increased phosphorylation of Akt in liver, and improved liver insulin resistance.