Tribbles Homolog 3 mRNA Research Articles

A human-specific VNTR in the TRIB3 promoter causes gene expression variation between individuals.

Tribbles homolog 3 (TRIB3) is pseudokinase involved in intracellular regulatory processes and has been implicated in several diseases. In this article, we report that human TRIB3 promoter contains a 33-bp variable number tandem repeat (VNTR) and characterize the heterogeneity and function of this genetic element. Analysis of human populations around the world uncovered the existence of alleles ranging from 1 to 5 copies of the repeat, with 2-, 3- and 5-copy alleles being the most common but displaying considerable geographical differences in frequency. The repeated sequence overlaps a C/EBP-ATF transcriptional regulatory element and is highly conserved, but not repeated, in various mammalian species, including great apes. The repeat is however evident in Neanderthal and Denisovan genomes. Reporter plasmid experiments in human cell culture reveal that an increased copy number of the TRIB3 promoter 33-bp repeat results in increased transcriptional activity. In line with this, analysis of whole genome sequencing and RNA-Seq data from human cohorts demonstrates that the copy number of TRIB3 promoter 33-bp repeats is positively correlated with TRIB3 mRNA expression level in many tissues throughout the body. Moreover, the copy number of the TRIB3 33-bp repeat appears to be linked to known TRIB3 eQTL SNPs as well as TRIB3 SNPs reported in genetic association studies. Taken together, the results indicate that the promoter 33-bp VNTR constitutes a causal variant for TRIB3 expression variation between individuals and could underlie the results of SNP-based genetic studies.

Tribbles Homolog 3 (TRB3) Expression is Modulated During Skeletal Muscle Myogenesis and Regeneration

Skeletal muscle mass and activity are primordial for homeostasis and are related to important functions such as breathing, locomotion and glucose metabolism. This tissue is susceptible to lesion from mechanical‐chemical injuries and also from the mechanical stress generated by contractions. Fortunately, skeletal muscle is highly capable of regeneration, although the cellular‐molecular mechanisms involved are still not properly understood. TRB3 is a gene that codes the homonymous protein often studied for its involvement in insulin resistance by inhibiting Akt2 activity. Because it has also been shown that TRB3 overexpression can decrease myotube formation, we decided to evaluate the TRB3 expression during skeletal muscle regeneration and also in myogenesis using C2C12‐derived myotubes as a model. We cultured C2C12 cells using DMEM 10% FBS with antibiotics as growth medium. Cells at ~80% confluence were induced to differentiation into myotubes with DMEM 2% for 2 and 4 days. Cells were then harvested and total RNA and protein were isolated for further analysis by using RT‐PCR and Western blot. Also, 16 adult male C57Bl mice (~8 weeks, ~30g) were housed in 12/12h light/dark regimen with food and water ad libitum and divided into Control and tibialis anterior‐injured by cardiotoxin (CTX, 10uM) for 1, 3 and 10 days. Statistical analyzes were conducted using t‐student test in comparison with Control Group (not injured mice) or one‐way ANOVA for nonparametric data with Dunns post‐hoc for C2C12 samples analysis, expressed in Mean +− SD and p≤0.05. All experiments were approved by the Institute of Biomedical Sciences Animal Experimentation Office Committee (6085260718). We observed elevated levels of TRB3 mRNA at 2 and 4 days of C2C12 differentiation (~2.5 fold for both time points, p<0.05). Interestingly, in spite of a clear rise in TRB3 mRNA, AKT2 (a target for TRB3) mRNA is also upregulated in differentiated C2C12 cells (~3.5 fold 4 days after differentiation, p<0.05). Likewise, GSK3b mRNA levels are elevated 4 days after differentiation (2.5 fold, p<0.05). In the regeneration model we also observed increased TRB3 mRNA levels in all time points evaluated (1 day~19 fold 3 days~9 fold and 10 days~5 fold, p<0.05). Accordingly, we preliminarily observed increased levels of TRB3 10 days after injury by Western blot. These results suggests that TRB3 might be involved in the early and late phases of the myogenic‐regenerative process and functional experiments involving manipulated TRB3 levels are ongoing.Support or Funding InformationFAPESP, CAPES, CNPq

TRB3 interacts with ERK and JNK and contributes to the proliferation, apoptosis, and migration of lung adenocarcinoma cells.

Tribbles homolog 3 (TRB3) has been accounted for regulation of a few cell processes through interaction with other significant proteins. The molecular mechanisms underlying TRB3 in tumorigenesis in lung adenocarcinoma have not been entirely elucidated. The present study is aimed at determining the function and fundamental mechanisms of TRB3 in lung adenocarcinoma progression. TRB3 was highly expressed in A549 and H1299 cells and lung adenocarcinoma tissues compared with human bronchial epithelial cells (HBEpC) and adjacent normal lung tissues. Hypoxia significantly upregulated the expression of TRB3 protein in A549 and H1299 cells in a time-dependent way. Gene expression profiling interactive analysis data analysis indicated that patients withlung adenocarcinoma with excessive expression of TRB3 mRNA had fundamentally shorter survival time. TRB3 knockdown in A549 cells can inhibit cell proliferation and migration, and promote cell apoptosis. TRB3 knockdown reduced the expression of p-ERK and p-JNK, but did not affect the expression of p-P38 MAPK. TRB3 overexpression enhances the malignant transformation abilities of HBEpC such as cell proliferation, migration and colony formation, which could be reversed by U0126 and SP600125. TRB3 overexpression promotes the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but was not affected by U0126 and SP600125. The results of coimmunoprecipitation experiments indicated that TRB3 binds directly to ERK and JNK. This study suggests that TRB3 has a potentially carcinogenic role in lung adenocarcinoma by binding to ERK and JNK and promoting the phosphorylation of ERK and JNK. TRB3 can be a possible therapeutic focus for lung adenocarcinoma.

TRIB3 enhances cell viability during glucose deprivation in HEK293-derived cells by upregulating IGFBP2, a novel nutrient deficiency survival factor

Glucose deprivation occurs in several human diseases, including infarctions and solid tumors, and leads to cell death. In this article, we investigate the role of the pseudokinase Tribbles homolog 3 (TRIB3) in the cellular stress response to glucose starvation using cell lines derived from HEK293, which is highly glycolytic under standard conditions. Our results show that TRIB3 mRNA and protein levels are strongly upregulated in glucose-deprived cells via the induction of activating transcription factor 4 (ATF4) by the endoplasmic reticulum (ER) stress sensor kinase PERK. Cell survival in glucose-deficient conditions is enhanced by TRIB3 overexpression and reduced by TRIB3 knockdown. Genome-wide gene expression profiling uncovered approximately 40 glucose deprivation-responsive genes that are affected by TRIB3, including several genes involved in signaling processes and metabolism. Based on transcription factor motif analysis, the majority of TRIB3-downregulated genes are target genes of ATF4, which TRIB3 is known to inhibit. The gene most substantially upregulated by TRIB3 is insulin-like growth factor binding protein 2 (IGFBP2). IGFBP2 mRNA and protein levels are downregulated in cells subjected to glucose deprivation, and reduced IGFBP2 expression aggravates cell death during glucose deficiency, while overexpression of IGFBP2 prolongs cell survival. Moreover, IGFBP2 silencing abrogates the pro-survival effect of TRIB3. Since TRIB3 augments IGFBP2 expression in glucose-starved cells, the data indicate that IGFBP2 contributes to the attenuation of cell death by TRIB3. These results implicate TRIB3 and IGFBP2, both of which are known to be overexpressed in several types of cancers, as pro-survival modulators of cell viability in nutrient-deficient microenvironments.

Tribbles homolog 3 is induced by high glucose and associated with apoptosis in human endothelial cells.

Tribbles homolog 3 (TRIB3) is an intracellular kinase-like molecule that modifies cellular survival and metabolism. The present study aimed to investigate the function of TRIB3 regulation in the process of high glucose-induced apoptosis in endothelial cells, with the aim of identifying a novel intervention target for the prevention and treatment of diabetes mellitus. Human umbilical vein endothelial cells (HUVECs) grown in medium with various concentrations of glucose (5.5, 10, 20, 30 and 40 mmol/l) were assessed for mRNA expression of TRIB1, TRIB2 and TRIB3 using reverse transcription quantitative polymerase chain reaction. In addition, protein expression of TRIB3 was examined using western blot analysis. Immunofluorescence staining was performed in order to determine the distribution and localization of TRIB3 in HUVECs. Furthermore, cells grown in normal (5.5 mmol/l) or high glucose (HG; 30 mmol/l) medium were subjected to TRIB3 inhibition through small interfering (si)RNA knockdown. These cells were then examined in order to determine whether TRIB3 upregulation was associated with endothelial cell apoptosis. HUVECs treated with 30 and 40 mmol/l glucose for 48 h and 72 h showed significantly lower survival rates compared with those treated with normal glucose levels. In addition, slight but not significant increases in TRIB1 and TRIB2 mRNA expression were observed in HUVECs incubated with various concentrations of glucose for different durations. By contrast, TRIB3 mRNA expression was increased 7.2-fold following incubation with HG. Western blot analysis revealed a 5.44-fold increase in TRIB3 protein levels in cells grown in HG medium for 24 h compared with those grown in normal medium. Immunostaining assays revealed a markedly higher and well-defined nucleolar fluorescence intensity for TRIB3 expression at 24 h in HG medium compared with that of the control group. Furthermore, the apoptotic rate of HG-treated TRIB3 siRNA-transfected HUVECs was significantly increased compared with that of those transfected with control siRNA In conclusion, the results of the present study suggested that TRIB3 was associated with high glucose-induced HUVECs apoptosis, which was attenuated following transfection with TRIB3 siRNA.

Regulation of TRIB3 mRNA and Protein in Breast Cancer

Tribbles homolog 3 (TRIB3) is a scaffold protein activated under hypoxic conditions and involved in several cell survival and proliferation pathways. Recently, we reported opposite associations of TRIB3 mRNA and protein with breast cancer prognosis. In this study, we investigated this discrepancy between TRIB3 mRNA and protein in human breast cancer. We provide several lines of evidence demonstrating that TRIB3 is a stabile protein which levels are not controlled by rapid protein breakdown. Interestingly, we were able to show that during anoxia TRIB3 mRNA translation was profoundly inhibited. Hypoxia induced micro RNA 24 was not responsible for the translational repression of TRIB3. Furthermore miRNA-24 expression levels in breast cancer patient specimens showed no correlation with TRIB3 mRNA or TRIB3 protein levels, or with prognosis. Thus, the expression of miRNA-24 does not explain the difference between mRNA and protein expression of TRIB3 in this cohort of breast cancer patients. In conclusion, TRIB3 protein is a stable protein which levels are predominantly regulated by translational control of TRIB3 mRNA transcript.

Role of TRIB3 in regulation of insulin sensitivity and nutrient metabolism during short-term fasting and nutrient excess

We have suggested previously that Tribbles homolog 3 (TRIB3), a negative regulator of Akt activity in insulin-sensitive tissues, could mediate glucose-induced insulin resistance in muscle under conditions of chronic hyperglycemia (Liu J, Wu X, Franklin JL, Messina JL, Hill HS, Moellering DR, Walton RG, Martin M, Garvey WT. Am J Physiol Endocrinol Metab 298: E565-E576, 2010). In the current study, we have assessed short-term physiological regulation of TRIB3 in skeletal muscle and adipose tissues by nutrient excess and fasting as well as TRIB3's ability to modulate glucose transport and mitochondrial oxidation. In Sprague-Dawley rats, we found that short-term fasting enhanced insulin sensitivity concomitantly with decrements in TRIB3 mRNA (66%, P < 0.05) and protein (81%, P < 0.05) in muscle and increments in TRIB3 mRNA (96%, P < 0.05) and protein (~10-fold, P < 0.05) in adipose tissue compared with nonfasted controls. On the other hand, rats fed a Western diet for 7 days became insulin resistant concomitantly with increments in TRIB3 mRNA (155%, P < 0.05) and protein (69%, P = 0.0567) in muscle and a decrease in the mRNA (76%, P < 0.05) and protein (70%, P < 0.05) in adipose. In glucose transport and mitochondria oxidation studies using skeletal muscle cells, we found that stable TRIB3 overexpression impaired insulin-stimulated glucose uptake without affecting basal glucose transport and increased both basal glucose oxidation and the maximal uncoupled oxygen consumption rate. With stable knockdown of TRIB3, basal and insulin-stimulated glucose transport rates were increased, whereas basal glucose oxidation and the maximal uncoupled oxygen consumption rate were decreased. In conclusion, TRIB3 impacts glucose uptake and oxidation oppositely in muscle and fat according to levels of nutrient availability. The above data for the first time implicate TRIB3 as a potent physiological regulator of insulin sensitivity and mitochondrial glucose oxidation under conditions of nutrient deprivation and excess.

Study on the Protective Effect of Shengmai San ( ) on the Myocardium in the Type 2 Diabetic Cardiomyopathy Model Rat

To study the effect of Shengmai San ((see text) Pulse-activating Powder) in protecting myocardium in the rat of the type 2 diabetic cardiomyopathy (DCM) model. The DCM rat model was established by combination of insulin resistance induced by a high-fat diet with intraperitoneal injection of high dose streptozotocin (50 mg/kg). And these rat models were randomly divided into three groups: a normal group (n = 12,one of them died), a model group (n = 15) and a Shengmai San group (treatment group, n = 15).The damage of the myocardium was assessed by electrocardiogram at the twelfth week after modeling, and the blood glucose, cholesterol and triglyceride levels were determined; the content of the left cardiac ventricle myocardial collagen was quantified by Masson staining test; the level of myocardial cell apoptosis was detected with TUNEL apoptosis detection kit; the damage extent of the myocardial sub-cellular structures was observed by electron microscopy; the expression levels of cardiac TSP-1 (Thrombospondin-1), TGF-beta1 (Transforming Growth F factor-beta) and TRB-3 (Tribbles homolog 3) proteins were detected by immunohistochemical method; the expression levels of cardiac TSP-1, A-TGF-beta1 and L-TGF-beta1 proteins were detected by Western blotting; and the expression levels of TSP-1 and TRB-3 mRNAs were detected by real-time quantitative PCR. Compared with the control group, the blood glucose, cholesterol, triglycerides levels in both the model groups and the Shengmai San group were significantly decreased; the myocardial tissue was less damaged and the collagen content was reduced in the Shengmai San group; the myocardial sub-cellular structure was injured to a lesser extent; the expression levels of myocardial TSP-1, TGF-beta1, TRB-3, and TSP-1, A-TGF-beta1, L-TGF-beta1 and chymase were decreased, and the expression levels of TSP-1 mRNA and TRB-3 mRNA were decreased in both the model groups and the Shengmai San group (the latter was better),. Shengmai San can inhibit myocardial fibrosis in the rat of diabetic cardiomyopathy, and significantly delay the formation of diabetic cardiomyopathy in hyperglycemia rats through multiple pathways.

Study on the protective effect of the Mixture of Shengmai Powder and Danshen Decoction (生脉散丹参饮合剂) on the myocardium of diabetic cardiomyopathy in the rat model

To study the protective effect of the Mixture of Shengmai Powder and Danshen Decoction (, abbreviated as the Mixture) in the rat model with type 2 diabetic cardiomyopathy in the rat model with type 2 diabetic cardiomyopathy, abbreviated as the Mixture) in the rat model with type 2 diabetic cardiomyopathy (DCM). Forty-two SD rats with DCM model, established by the combination of insulin resistance by a high-fat diet with the damage of pancreatic islet β cells by intraperitoneal injection of high dose streptozotocin (50 mg/kg) once, were evaluated in the damage of the myocardium by electrocardiogram at the end of 12 weeks of grouping and intervention administration; the extent of damage in the myocardial subcellular structure was observed by electron microscopy; the content of myocardial collagen in the left cardiac ventricle was quantified by Masson staining test; the myocardial cell apoptosis was determined by TUNEL; the changes in the mRNA expression levels of thrombospodin-1 (TSP-1) and tribbles homolog 3 (TRB-3) by real-time quantitative PCR, the expression levels of myocardial TSP-1, tumor growth factorβ1 (TGF-β1), TRB-3, and chymase were detected by immunohistochemistry, and the changes in the expression levels of myocardial TSP-1, active-TGF-β1 (A-TGF-β1) and latent-TGF-β1 (L-TGF-β1) protein were tested by Western blotting. Compared with the control group, the myocardial tissue was less damaged, and the extent of damage in the myocardial subcellular structure was less; the collagen fiber content and the cell apoptosis were reduced; the expression levels of TSP-1mRNA and TRB-3 mRNA, the expression levels of myocardial TSP-1, TGF-β1, TRB-3, and chymase, as well as the average expression levels of the myocardial TSP-1, A-TGFβ1, and L-TGF-β1 protein were decreased in the Mixture group. The Mixture of Shengmai Powder and Danshen Decoction could inhibit the process of myocardial fibrosis in the rat myocardium of DCM through multiple pathways and significantly delay the genesis and progress of DCM in hyperglycemic rats.

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

Mammalian Tribbles homolog 3 impairs insulin action in skeletal muscle: role in glucose-induced insulin resistance.

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes.

Human TRB3 is upregulated in stressed cells by the induction of translationally efficient mRNA containing a truncated 5′-UTR

Tribbles homolog 3 (TRB3) is a pseudokinase that has been implicated in the control of stress response, cell viability and metabolic processes, and has been linked to medical conditions, including insulin resistance, cardiovascular disease and diabetes. Therefore, the understanding of mechanisms that regulate TRB3 expression is of considerable importance. We have previously described the existence of several human (h) TRB3 mRNA isoforms that differ in their 5′-untranslated region (5′-UTR). In this study, we use a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) system to characterize the expression levels of hTRB3 mRNA isoforms in HepG2 hepatoma cells cultured in regular medium or exposed to arsenite, and investigate the effect of hTRB3 5′-UTR variants on the efficiency of mRNA translation. The data indicate that of the hTRB3 mRNA splice variants, 1A is predominant (> 80% of molecules) in both the stressed and unstressed states, and that the remainder consists mainly of 1B4, with the variants 1B1, 1B2 and 1B3 together forming less than 1% of the population in either condition. In addition to the substantial transcriptional upregulation of all hTRB3 mRNA splice variants, the exposure of cells to arsenite results in a marked increase in the proportion of splice variant 1A molecules containing a truncated 5′-UTR. The shortened 1A 5′-UTR proved to be translationally more efficient than the untruncated 1A 5′-UTR, due to the lack of an inhibitory upstream open reading frame (uORF). Thus, increased transcription as well as altered usage of 5′-UTR variants contributes to the upregulation of hTRB3 protein synthesis in stressful conditions.