TRIB3 Research Articles

The proliferation and differentiation of spermatogonial stem cells in the frist wave of spermatogenesis in rats with Trib3 gene knockout

This study explores the effects of Trib3 gene knockout on adult male rat spermatogenesis. Using CRISPR/Cas9, we knocked out the Trib3 gene in Wistar rats. Results indicate altered expression of PLZF, ID4, and c-KIT in knockout rats, suggesting impaired spermatogonial stem cell proliferation and differentiation. Histological analysis reveals reduced seminiferous tubule area and decreased spermatocyte numbers. Mating experiments demonstrate reduced offspring rates after the second self-mating in knockout rats. SYCP3, a meiosis marker, shows elevated expression in knockout rat testes at 14 days postpartum, suggesting an impact on reproductive processes. ELISA results indicate decreased testosterone, FSH, and FGF9 levels in knockout rat testicular tissues. In conclusion, Trib3 gene deletion may impede spermatogonial self-renewal and promote differentiation through the FSH-FGF9- c-KIT interaction and p38MAPK pathway, affecting reproductive capacity. These findings contribute to understanding the molecular mechanisms regulating spermatogenesis.

Exudative Age-Related Macular Degeneration: Association between Treatment Efficacy and Single-Nucleotide Variants in RAD51B, TRIB1, COL8A1, COL10A1, IL-9, IL-10, and VEGFA Genes.

Age-related macular degeneration (AMD) is a progressive neurodegenerative condition leading to vision loss and eventual blindness, with exudative AMD posing a heightened risk due to choroidal neovascularization and localized edema. Therapies targeting the VEGF pathway aim to address this mechanism for treatment effectiveness. Our study aimed to evaluate associations between specific genetic variants (RAD51B rs8017304, rs2588809; TRIB1 rs6987702, rs4351379; COL8A1 rs13095226; COL10A1 rs1064583; IL-9 rs1859430, rs2069870, rs11741137, rs2069885, rs2069884; IL-10 rs1800871, rs1800872, rs1800896; VEGFA rs1570360, rs699947, rs3025033, rs2146323) and the response to anti-VEGF treatment for exudative AMD. We enrolled 119 patients with exudative AMD categorized as responders or non-responders based on their response to anti-VEGF treatment. Statistical analysis revealed that RAD51B rs8017304 heterozygous and homozygous minor allele carriers had increased CMT before treatment compared to wild-type genotype carriers (p = 0.004). Additionally, TRIB1 rs4351379 heterozygous and homozygous minor allele carriers exhibited a greater decrease in central macular thickness (CMT) after 6 months of treatment than wild-type genotype carriers (p = 0.030). IL-9 rs1859430, rs2069870, and rs2069884 heterozygous and homozygous minor allele carriers had worse BCVA before treatment than wild-type genotype carriers (p = 0.018, p = 0.012, p = 0.041, respectively). Conversely, IL-9 rs2069885 heterozygous and homozygous minor allele carriers showed greater improvement in BCVA after 6 months compared to wild-type genotype carriers (p = 0.032). Furthermore, VEGFA rs699947 heterozygous and homozygous minor allele carriers had better BCVA before treatment and after 3 and 6 months of treatment than wild-type genotype carriers (p = 0.003, p = 0.022, respectively), with these carriers also exhibiting higher CMT after 6 months of anti-VEGF treatment (p = 0.032). Not all results remained statistically significant under this stringent correction for multiple comparisons. The comparisons of the serum concentrations of IL-10, VEGF-A, and VEGF-R2/KDR between non-responders and responders did not yield statistically significant differences. Our study identified significant associations between genetic variants, including RAD51B rs8017304, TRIB1 rs4351379, IL-9 rs1859430, rs2069870, rs2069884, rs2069885, and VEGFA rs699947, and parameters related to the efficacy of exudative AMD treatment, such as BCVA and CMT.

TRIB1 confers therapeutic resistance in GBM cells by activating the ERK and Akt pathways

GBM (Glioblastoma) is the most lethal CNS (Central nervous system) tumor in adults, which inevitably develops resistance to standard treatments leading to recurrence and mortality. TRIB1 is a serine/threonine pseudokinase which functions as a scaffold platform that initiates degradation of its substrates like C/EBPα through the ubiquitin proteasome system and also activates MEK and Akt signaling. We found that increased TRIB1 gene expression associated with worse overall survival of GBM patients across multiple cohorts. Importantly, overexpression of TRIB1 decreased RT/TMZ (radiation therapy/temozolomide)-induced apoptosis in patient derived GBM cell lines in vitro. TRIB1 directly bound to MEK and Akt and increased ERK and Akt phosphorylation/activation. We also found that TRIB1 protein expression was maximal during G2/M transition of cell cycle in GBM cells. Furthermore, TRIB1 bound directly to HDAC1 and p53. Importantly, mice bearing TRIB1 overexpressing tumors had worse overall survival. Collectively, these data suggest that TRIB1 induces resistance of GBM cells to RT/TMZ treatments by activating the cell proliferation and survival pathways thus providing an opportunity for developing new targeted therapeutics.

Trib1 Deficiency Promotes Hyperlipidemia, Inflammation, and Atherosclerosis in LDL Receptor Knockout Mice.

Genetic variants at the TRIB1 gene locus are strongly associated with plasma lipid traits and the risk of coronary artery disease in humans. Here, we analyzed the consequences of Trib1 deficiency on lipid metabolism and atherosclerotic lesion formation in atherosclerosis-susceptible Ldlr-/- mice. Trib1-/- mice were crossed onto the Ldlr-/- background to generate double-knockout mice (Trib1-/-Ldlr-/-) and fed a semisynthetic, modified AIN76 diet (0.02% cholesterol and 4.3% fat) until 20 weeks of age. Trib1-/-Ldlr-/- mice had profoundly larger (5.8-fold) and more advanced atherosclerotic lesions at the aortic root as compared with Trib1+/+Ldlr-/- controls. Further, we observed significantly elevated plasma total cholesterol and triglyceride levels in Trib1-/-Ldlr-/- mice, resulting from higher VLDL (very-low-density lipoprotein) secretion. Lipidomics analysis revealed that loss of Trib1 altered hepatic lipid composition, including the accumulation of cholesterol and proinflammatory ceramide species, which was accompanied by signs of hepatic inflammation and injury. Concomitantly, we detected higher plasma levels of IL (interleukin)-6 and LCN2 (lipocalin 2), suggesting increased systemic inflammation in Trib1-/-Ldlr-/- mice. Hepatic transcriptome analysis demonstrated significant upregulation of key genes controlling lipid metabolism and inflammation in Trib1-/-Ldlr-/- mice. Further experiments suggested that these effects may be mediated through pathways involving a C/EPB (CCAAT/enhancer binding protein)-PPARγ (peroxisome proliferator-activated receptor γ) axis and JNK (c-Jun N-terminal kinase) signaling. We provide experimental evidence that Trib1 deficiency promotes atherosclerotic lesion formation in a complex manner that includes the modulation of lipid metabolism and inflammation.

HERV-K (HML-2) insertion polymorphisms in the 8q24.13 region and their potential etiological associations with acute myeloid leukemia.

Human endogenous retroviruses (HERVs) are LTR retrotransposons that are present in the human genome. Among them, members of the HERV-K (HML-2) group are suspected to play a role in the development of different types of cancer, including lung, ovarian, and prostate cancer, as well as leukemia. Acute myeloid leukemia (AML) is an important disease that causes 1% of cancer deaths in the United States and has a survival rate of 28.7%. Here, we describe a method for assessing the statistical association between HERV-K (HML-2) transposable element insertion polymorphisms (or TIPs) and AML, using whole-genome sequencing and read mapping using TIP_finder software. Our results suggest that 101 polymorphisms involving HERV-K (HML-2) elements were correlated with AML, with a percentage between 44.4 to 56.6%, most of which (70) were located in the region from 8q24.13 to 8q24.21. Moreover, it was found that the TRIB1, LRATD2, POU5F1B, MYC, PCAT1, PVT1, and CCDC26 genes could be displaced or fragmented by TIPs. Furthermore, a general method was devised to facilitate analysis of the correlation between transposable element insertions and specific diseases. Finally, although the relationship between HERV-K (HML-2) TIPs and AML remains unclear, the data reported in this study indicate a statistical correlation, as supported by the χ2 test with p-values < 0.05.

Direct-to-Consumer Genetic Test and Lifestyle Questionnaire Analysis of Body Mass Index and Body Fat Percentage in a Large Korean Population

Background: Direct-to-Consumer (DTC) genetic testing provides genetic risk to consumers and motivates consumers to take care of their own customized health care. In 2018, we developed and provided a DTC genetic testing service (GENESTART™) in collaboration with Herbalife Korea Co. Ltd. Methods: The analyzed dataset consisted of the body fat percentage (BFP), body mass index (BMI), 31 genetic polymorphism genotypes, and responses to 19 questionnaire items of 24,447 individuals. The genetic main effects for BFP and BMI were examined by linear regression analysis, and the interaction effects were examined using a generalized linear model that controlled age and sex as covariates. Results: In the case of BFP, the sample average was 31.47% overall, 24.76% for men, and 32.79% for women, showing that men had an average BFP that was 8 percentage points lower than that of women. The average BMI was 25.38 overall, 26.45 for men, and 25.17 for women, showing that men had an average BMI of 1.2 kg/m2 higher than that of women. The FTO and MC4R genes, well-known obesity markers, showed a significant correlation with both phenotypes, and the BDNF gene, which is related to stress obesity, showed a highly significant association with BMI but only a weak association with BFP. Among the remaining genes, TRIB1, ABCA1, MYL2, G6PC, GCKR, GLIS3, CYP17A1, HECTD4, and NT5C2 genes showed significant associations with the obesity-related phenotypes. In this study, we found four interaction results for BFP (ABO and fruits, CYP1A2 and sugary foods, FTO and muscle exercise, MC4R and vitamins) and five interactions for BMI (MC4R and proteins, CSK and fruits, MC4R and calcium, DGKB and calcium, CSK and water). Conclusions: This study is expected to enable the provision of personalized and accurate solutions for BFP and BMI management to customers who have undergone genetic testing.

Effect of TRIB1 Variant on Lipid Profile and Coronary Artery Disease: A Systematic Review and Meta-Analysis.

Emerging evidence indicates tribbles homolog 1 (Trib1) protein may be involved in lipid metabolism regulation and coronary artery disease (CAD) pathogenesis. However, whether TRIB1 gene variants affect lipid levels and CAD remains elusive, this study is aimed at clarifying the effect of TRIB1 variants on lipid profile and CAD. By searching PubMed and Cochrane databases for studies published before December 18, 2022, a total of 108,831 individuals were included for the analysis. The outcomes of the analysis on all individuals showed that the A allele carriers of rs17321515 and rs2954029 variants had higher low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) levels than the noncarriers. Consistently, a higher CAD risk was observed in the A allele carriers. Subgroup analysis indicated that increased LDL-C, TC, and CAD risk were observed in Asian population. Variants of TRIB1 (i.e., rs17321515 and rs2954029) may serve as causal genetic markers for dyslipidemia and CAD in Asian population.

Molecular characterization of TRIB1 gene and its role in regulation of steroidogenesis in bos grunniens granulosa cells

To explore the expression pattern of the TRIB1 gene in yak follicles and its effect on the steroidogenesis of granulosa cells (GCs). Here, 4–5 years old female yaks were treated as the subjects. Immunohistochemically assay found that TRIB1 protein was expressed in different developmental follicles. Among different cell types of follicles, including cumulus cells (CCs), granulosa cells (GCs) and theca cells (TCs), the TRIB1 protein was most abundant in GCs (P < 0.0001). In addition, we cloned the coding sequence (CDS) of the yak TRIB1 gene, which is 1119 bp, encoding 372 amino acids (AA). The amino acid sequence homology of TRIB1 is >80% to those of other species, except for zebrafish. To further explore the function of TRIB1 in steroidogenesis, the pcDNA3.1(+)-TRIB1 eukaryotic expression vector was constructed and then transfected into GCs. The data showed that overexpression of TRIB1 significantly reduced the progesterone (P4) secretion of granulosa cells measured by ELISA assay (P < 0.05), but not Estradiol (E2) secretion. Consistently, TRIB1 gain-of-function downregulated the mRNA levels of steroidogenesis related genes steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and 3β-hydroxysteroid dehydrogenase (3β-HSD) (P < 0.01), while cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) had no significant difference (P > 0.05). Interestingly, mito-tracker staining showed that mitochondrial number significantly decreased in TRIB1 overexpressed GCs (P < 0.01). Further, overexpression of TRIB1 inhibited mRNA levels of mitochondrial biogenesis related genes, including Mitochondrial transcription factor (TFAM) and Peroxisome proliferator-activated receptor alpha co-activator (PPARGC1A) (P < 0.05). Conclusively, this work indicates that TRIB1 inhibited progesterone synthesis of GCs might be involved in the reduction of the mitochondria number.

Machine learning enables new insights into genetic contributions to liver fat accumulation

Excess liver fat, called hepatic steatosis, is a leading risk factor for end-stage liver disease and cardiometabolic diseases but often remains undiagnosed in clinical practice because of the need for direct imaging assessments. We developed an abdominal MRI-based machine-learning algorithm to accurately estimate liver fat (correlation coefficients, 0.97-0.99) from a truth dataset of 4,511 middle-aged UK Biobank participants, enabling quantification in 32,192 additional individuals. 17% of participants had predicted liver fat levels indicative of steatosis, and liver fat could not have been reliably estimated based on clinical factors such as BMI. A genome-wide association study of common genetic variants and liver fat replicated three known associations and identified five newly associated variants in or near the MTARC1, ADH1B, TRIB1, GPAM, and MAST3 genes (p < 3 × 10-8). A polygenic score integrating these eight genetic variants was strongly associated with future risk of chronic liver disease (hazard ratio > 1.32 per SD score, p < 9 × 10-17). Rare inactivating variants in the APOB or MTTP genes were identified in 0.8% of individuals with steatosis and conferred more than 6-fold risk (p < 2 × 10-5), highlighting a molecular subtype of hepatic steatosis characterized by defective secretion of apolipoprotein B-containing lipoproteins. We demonstrate that our imaging-based machine-learning model accurately estimates liver fat and may be useful in epidemiological and genetic studies of hepatic steatosis.

TRIB1 regulates LDL metabolism through CEBPα-mediated effects on the LDL receptor in hepatocytes.

Genetic variants near the TRIB1 gene are highly significantly associated with plasma lipid traits and coronary artery disease. While TRIB1 is likely causal of these associations, the molecular mechanisms are not well understood. Here we sought to investigate how TRIB1 influences low density lipoprotein cholesterol (LDL-C) levels in mice. Hepatocyte-specific deletion of Trib1 (Trib1Δhep) in mice increased plasma cholesterol and apoB and slowed the catabolism of LDL-apoB due to decreased levels of LDL receptor (LDLR) mRNA and protein. Simultaneous deletion of the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα) with TRIB1 eliminated the effects of TRIB1 on hepatic LDLR regulation and LDL catabolism. Using RNA-seq, we found that activating transcription factor 3 (Atf3) was highly upregulated in the livers of Trib1Δhep but not Trib1Δhep CebpaΔhep mice. ATF3 has been shown to directly bind to the CEBPα protein, and to repress the expression of LDLR by binding its promoter. Blunting the increase of ATF3 in Trib1Δhep mice reduced the levels of plasma cholesterol and partially attenuated the effects on LDLR. Based on these data, we conclude that deletion of Trib1 leads to a posttranslational increase in CEBPα, which increases ATF3 levels, thereby contributing to the downregulation of LDLR and increased plasma LDL-C.

Tribbles Pseudokinase 3 Regulation and Contribution to Cancer.

Simple SummaryAccumulating evidence supports a key function for Tribbles proteins in oncogenesis, both in leukemia and solid tumors. However, the exact role of these proteins is hard to define since in a context-dependent manner they can function as both oncogenes and tumor suppressors. Their complex role arises from the capacity to interact with a wide range of target molecules thereby acting as molecular scaffolds and signaling regulators of multiple pathways. This review focuses on one particular Tribbles family member, namely, TRIB3, addressing its gene and protein expression, as well as its role in cancer development and progression.The first Tribbles protein was identified as critical for the coordination of morphogenesis in Drosophila melanogaster. Three mammalian homologs were subsequently identified, with a structure similar to classic serine/threonine kinases, but lacking crucial amino acids for the catalytic activity. Thereby, the very weak ATP affinity classifies TRIB proteins as pseudokinases. In this review, we provide an overview of the regulation of TRIB3 gene expression at both transcriptional and post-translational levels. Despite the absence of kinase activity, TRIB3 interferes with a broad range of cellular processes through protein–protein interactions. In fact, TRIB3 acts as an adaptor/scaffold protein for many other proteins such as kinase-dependent proteins, transcription factors, ubiquitin ligases, or even components of the spliceosome machinery. We then state the contribution of TRIB3 to cancer development, progression, and metastasis. TRIB3 dysregulation can be associated with good or bad prognosis. Indeed, as TRIB3 interacts with and regulates the activity of many key signaling components, it can act as a tumor-suppressor or oncogene in a context-dependent manner.

Improving the Effects of Omega-3 Fatty Acid on the In Vitro Maturation of Oocytes

This research was conducted in order to determine the effects of omega-3 on oocyte in vitro maturation and the level of expression of tribbles (TRIB1, TRIB2 and TRIB3 genes) in cumulus cells. Eight-ten weeks old NMRI mice were super-ovulated using 7.5 IU pregnant mare’s serum gonadotropin (PMSG, Intraperitoneal) and they were killed after 44 hours and their ovaries were removed. The oocytes were used for &lt;em&gt;in vitro&lt;/em&gt; maturation and the cumulus-oocyte complexes (COCs) were released. Cumulus cells and oocytes were assigned into control, ethanol-treated and groups exposed to 10 and 100 μg/ml of omega-3. The cells were prepared to assess the maturation stage in order to evaluate the gene expression level. The data were statistically analyzed. Exposing oocytes to low dose (10 μg/ml) and high dose (100 μg/ml) of omega-3 resulted in a reduced rate of GV-stage oocytes, decreased MI-oocytes and increased MII-oocytes. The enhanced maturity of COCs was also detected in response to a high dose of omega-3 (100 μg/ml). Exposure of cumulus cells to omega-3 (10 and 100 μg/ml) induced TRIB2 and inhibited TRIB3 gene expression level; however, TRIB1 gene expression level increased and decreased in response to low (10 μg/ml) and high (100 μg/ml) concentrations of omega-3, respectively. The addition of omega-3 to the environment of oocytes or cumulus cells affected the maturation of oocytes and cumulus cells, which was followed by the differential expression of TRIB genes, suggesting that there was a role of fatty acid metabolism in the differentiation and maturation of cumulus cells.

A Luminescent Eu(III) Coordination Polymer: Selective Detection of p-Nitroaniline and Treatment Effect on Colon Cancer by Inhibiting TRIB3 Mediated Cell Proliferation and Migration

By employment of the flexible bis-(imidazole-4,5-dicarboxylate) ligand (H4L) ligand, a new luminescent Eu(III)-containing coordination polymer {[Eu(HL)(H2O)2]·2(H2O)}n has been successfully synthesized under hydrothermal condition by reaction of Eu(NO3)3·6H2O with H4L with the presence of NaOH as the pH modulator. The chemical composing of the as-prepared complex 1 has been determined via the elemental analysis and the structural arrangement was probed via the single crystal X-ray diffraction. Furthermore, complex 1 could also be applied as a luminescent sensor for the detection of p-nitroaniline, which has high sensitivity and selectivity. In biological research, the related anti-colon cancer activity on SW480 cancer cells was further evaluated. Firstly, the effect of compound on TRIB3 gene expression was evaluated by RT-PCR. The CCK-8 and transwell assay were next be performed to detect the inhibitory function of compound on SW480 cancer cell proliferation and migration. Finally, the results obtained through molecular docking simulation shed light on the understanding of Eu(II) metallic complex on DNA.

TFAP2C Promotes Cell Proliferation by Upregulating CDC20 and TRIB3 in Non-small Cell Lung Cancer Cells

TFAP2C Promotes Cell Proliferation by Upregulating CDC20 and TRIB3 in Non-small Cell Lung Cancer Cells

RAD51B (rs8017304 and rs2588809), TRIB1 (rs6987702, rs4351379, and rs4351376), COL8A1 (rs13095226), and COL10A1 (rs1064583) Gene Variants with Predisposition to Age-Related Macular Degeneration.

Background Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of a central part of the neural retina (macula) and a leading cause of blindness in elderly people. While it is known that the AMD is a multifactorial disease, genetic factors involved in lipid metabolism, inflammation, and neovascularization are currently being widely studied in genome-wide association studies (GWAS). The aim of our study was to evaluate the impact of new single nucleotide polymorphisms (SNPs) in RAD51B, TRIB1, COL8A1, and COL10A1 genes on AMD development. Methods Case-control study involved 254 patients diagnosed with early AMD, 244 patients with exudative AMD, and 942 control subjects. The genotyping of RAD51B (rs8017304 and rs2588809), TRIB1 (rs6987702, rs4351379, and rs4351376), COL8A1 (rs13095226), and COL10A1 (rs1064583) was carried out using TaqMan assays by a real-time polymerase chain reaction (RT-PCR) method. Results Statistically significant difference was found in genotype (TT, TC, and CC) distribution of COL8A1 rs13095226 between exudative AMD and control groups (60.2%, 33.6%, and 6.1% vs. 64.9%, 32.3%, and 2.9%, respectively, p = 0.036). Also, comparing with TT+TC, rs13095226 CC genotype was associated with 3.5-fold increased odds of exudative AMD development (OR = 3.540; 95% CI: 1.415-8.856; p = 0.007). Conclusion Our study revealed a strong association between a variant in COL8A1 (rs13095226) and exudative AMD development.

TRIB1 rs17321515 and rs2954029 gene polymorphisms increase the risk of non-alcoholic fatty liver disease in Chinese Han population

BackgroundDysregulation of the lipid homeostasis is an independent risk factor for non-alcoholic fatty liver disease (NAFLD). Some studies had demonstrated that TRIB1 gene polymorphisms affect the plasma lipids metabolism, but no related data was available for TRIB1 gene polymorphisms in the lipids metabolism in Chinses Han population. The present study was conducted to investigate the association between TRIB1 gene polymorphisms (rs17321515 and rs2954029) and the risk of NAFLD in Chinese Han population and their effects on serum lipid profiles.Patients and methodsTRIB1 rs17321515 and rs2954029 gene polymorphisms were genotyped using the polymerase chain reaction (PCR) in B-type ultrasonography-proven NAFLD patients (n = 146) and healthy controls (n = 175). Serum lipid profiles were determined using biochemical methods. Statistical analyses were performed using SPSS 22.0 statistical software.ResultsThe allele distributions of TRIB1 rs17321515 A and rs2954029 A were significant different between the NAFLD patients and healthy controls (P = 0.026, P = 0.045, respectively). The genotype distribution of TRIB1 rs17321515 was significant different between NAFLD patients and healthy controls (P = 0.038). The TRIB1 rs17321515 GA + AA genotype and TRIB1 rs2954029 TA + AA genotype markedly increase the NAFLD risk (OR = 1.885; 95%CI: 1.157–3.070; OR = 1.627; 95%CI: 1.011–2.619, respectively), after adjusted for age, gender, and body mass index, the NAFLD risk still significant (OR = 2.240; 95%CI: 1.196–4.197; OR = 2.050; 95%CI: 1.110–3.786, respectively). In addition, TRIB1 rs17321515 A and rs2954029 A carriers possess the higher lipid profiles in the included subjects.ConclusionsTRIB1 rs17321515 and rs2954029 were significant associated with the risk of NAFLD in Chinese Han population. The rs17321515 A and rs2954029 A allele increases the serum lipid profiles in Chinese Han population.

Association Analysis of a Microsatellite Repeat in the TRIB1 Gene With Prostate Cancer Risk, Aggressiveness and Survival.

With an estimated 1.1 million men worldwide diagnosed with prostate cancer yearly, effective and more specific biomarkers for early diagnosis could lead to better patient outcome. As such, novel genetic markers are sought for this purpose. The tribbles homologue 1 gene (TRIB1) has recently shown to have a role in prostate tumorigenesis and data-mining of prostate cancer expression data confirmed clinical significance of TRIB1 in prostate cancer. For the first time, a polymorphic microsatellite in this gene was studied for its potential association with prostate cancer risk and aggressiveness. Genomic DNA was extracted from a cohort of 1,152 prostate cancer patients and 1,196 cancer-free controls and the TTTTG-TRIB1 microsatellite was genotyped. The socio-demographic and clinical characteristics were analyzed using the non-parametric t-test and two-way ANOVA. Association of the TTTTG-TRIB1 microsatellite and prostate cancer risk and aggressiveness were analyzed by binary logistic regression and confirmed by bootstrapping. Total and prostate cancer mortality was analyzed using the Kaplan Meier test. Genotype and allele correlation with TRIB1 mRNA levels was analyzed using the non-parametric Kolmogorov–Smirnov test. To predict the effect that the TTTTG-TRIB1 polymorphisms had on the mRNA structure, the in silico RNA folding predictor tool, mfold, was used. By analyzing the publicly available data, we confirmed a significant over-expression of TRIB1 in prostate cancer compared to other cancer types, and an over-expression in prostate cancerous tissue compared to adjacent benign. Three alleles (three–five repeats) were observed for TTTTG-TRIB1. The three-repeat allele was associated with prostate cancer risk at the allele (OR = 1.16; P = 0.044) and genotypic levels (OR = 1.70; P = 0.006) and this association was age-independent. The four-repeat allele was inversely associated with prosatet cancer risk (OR = 0.57; P < 0.0001). TRIB1 expression was upregulated in tumors when compared to adjacent cancer-free tissue but was not allele specific. In silico analysis suggested that the TTTTG-TRIB1 alleles may alter TRIB1 mRNA structure. In summary, the three-repeat allele was significantly associated with prostate cancer risk, suggesting a biomarker potential for this microsatellite to predict prostate cancer. Further studies are needed to elucidate the functional role of this microsatellite in regulating TRIB1 expression, perhaps by affecting the TRIB1 mRNA structure and stability.

The Induction of TRIB3 Causing Glucose Toxicity in Muscle Requires the O-GlcNAcylation of SP1

Background: Glucose-induced insulin resistance is mediated by induction of the pseudokinase TRIB3, which requires glucose metabolism via the hexosamine biosynthetic pathway (HBP). We have also shown that the SP1 binding site in the TRIB3 promoter region is essential for glucose’s ability to induce TRIB3 in skeletal muscle. However, the mechanism by which glucose regulates TRIB3 gene expression remains unknown. Hypothesis: Since the HBP provides the substrate for O-GlcNAcylation, we hypothesize that glucose results in O-GlcNAcylation of SP1 and this modulates induction of TRIB3 causing insulin resistance. Results: When treated with 5mM or 25mM glucose, the induction of TRIB3 gene expression (P&amp;lt;0.01) in L6 myotubes is accompanied by upregulation of SP1 expression (P&amp;lt;0.01). When fully differentiated L6 myotubes are transiently transfected with SP1 siRNA, knockdown of SP1 produced a corresponding suppression of TRIB3 both in the high and low glucose (P&amp;lt;0.05). To identify whether O-GlcNAcylation is involved, we cultured L6 myotubes in either 5mM or 25mM glucose medium with or without OSMI-1, an inhibitor of rate-limiting O-GlcNAc transferase (OGT). OSMI-1 did not affect glucose’s ability to augment SP1 2-fold (p&amp;lt;0.01), but effectively blocked induction of TRIB3. At the same time, OSMI-1 downregulated O-GlcNAcylation of SP1 and other O-GlcNAc modified proteins. Conclusion: (1) Increased TRIB3 mRNA expression parallels with upregulated SP1 expression; (2) Glucose induction of TRIB3 mRNA and protein is eliminated when HBP flux and O-GlcNAcylation is disrupted by the inhibition of OGT; (3) SP1 sites are critical in the regulatory action of glucose on the TRIB3 promoter and can be modulated by SP1 O-GlcNAcylation. While further studies are underway, these data provide new insights regarding mechanisms causing glucose-induced insulin resistance, and TRIB3 and TRIB3 promoter regulation are attractive targets for prevention of glucose toxicity in diabetes. Disclosure X. Liu: None. M. Kang: None. W. Garvey: Advisory Panel; Self; Novo Nordisk Inc., Merck &amp; Co., Inc.. Research Support; Self; Sanofi, Pfizer Inc., Novo Nordisk Inc., AstraZeneca, Merck &amp; Co., Inc., Elcelyx Therapeutics, Inc., Lexicon Pharmaceuticals, Inc., Eisai Inc..

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.

Tribbles Pseudokinase 3 Induces Both Apoptosis and Autophagy in Amyloid-β-induced Neuronal Death

Amyloid-β (Aβ)-induced neuron death is considered central to the pathogenesis of Alzheimer's disease (AD). Among several death modalities, autophagy and apoptosis play important roles in Aβ-induced neuron death suggesting that there may be regulatory mechanisms that initiate both cell death pathways. However, molecules that govern both pathways have not been identified. Here, we report that, upon Aβ treatment, tribbles pseudokinase 3 (Trib3, an ortholog of Drosophila Tribbles) is up-regulated in neurons both in vivo and in vitro Increased Trib3 levels inhibited the activity of the kinase Akt by interacting with it. As a result, forkhead box O1 (FoxO1), a transcription factor that is negatively regulated by Akt, was activated, translocated to the nucleus, and induced the pro-apoptotic gene BCL2-like 11 (Bim). Conversely, FoxO1 responded to Aβ insult by binding to the Trib3 gene promoter, enhancing its expression. Our investigations further revealed that Trib3 also induces autophagy. We found that Trib3 indirectly activates unc-51-like autophagy-activating kinase1 (Ulk1) by impeding phosphorylation of, and thus inactivating, a negative regulator of Ulk1, mechanistic target of rapamycin. Ulk1 activation augmented autophagosome formation and reduced autophagy flux. Thus, Trib3 was required for formation of autophagosomes, which accumulated in neurons as autophagic flux was thwarted. Most importantly, silencing endogenous Trib3 strongly protected neurons from Aβ insult. Our results suggest that a self-amplifying feed-forward loop among Trib3, Akt, and FoxO1 in Aβ-treated neurons induces both apoptosis and autophagy, culminating in neuron death. Thus, Trib3 may serve as a potential therapeutic target for AD.