TRIB3 Research Articles

P27 TRIB3: a regulator of metabolic syndromes and type 2 diabetes

Introduction The dys-regulation of multiple physiological systems, with obesity as a risk factor, contributes to various facets of metabolic diseases including diabetes, hypertension and cardiovascular disease. Trib-3 plays a role in many of these metabolic processes, including insulin resistance. The aim of this study was to understand the mechanisms by which trib-3 affects metabolism leading to obesity. Methods Trib-3-/- mouse was created using a gene-trap system thereby disrupting transcription of the Trib-3 mRNA. Microarray analysis was performed on Trib-3-/- and wild type littermate liver tissues. IPA software package was used for bioinformatics analysis of the data. qPCR and Western blots were carried out to substantiate the microarray findings. Tissue morphology was verified by histomorphometric analysis and triglyceride content was quantified using ELISA. Results Male Trib-3-/- mice are obese with elevated plasma levels of vLDL and cholesterol. These knockout mice also exert a fatty liver phenotype. Microarray analysis revealed the dysregulation of multiple metabolic genes including SAA1, PPAR-α and FCER1G with up-to 3x fold change. Western blots have confirmed reduced levels of PPAR-α (****P Conclusion A reduction in lipid-sensor PPARα suggest a molecular mechanisms for ‘mis-handling’ of fatty acids, causing accumulation in the liver leading to a dysfunctional, fatty liver. Our findings suggest that trib-3 plays an important part in regulating hepatic lipid homeostasis.

Co-operative leukemogenesis in acute myeloid leukemia and acute promyelocytic leukemia reveals C/EBPα as a common target of TRIB1 and PML/RARA.

The PML/RARA fusion protein occurs as a result of the t(15;17) translocation in the acute promyelocytic leukemia subtype of human acute myeloid leukemia. Gain of chromosome 8 is the most common chromosomal gain in human acute myeloid leukemia, including acute promyelocytic leukemia. We previously demonstrated that gain of chromosome 8-containing MYC is of central importance in trisomy 8, but the role of the nearby TRIB1 gene has not been experimentally addressed in this context. We have now tested the hypothesis that both MYC and TRIB1 have functional roles underlying leukemogenesis of trisomy 8 by using retroviral vectors to express MYC and TRIB1 in wild-type bone marrow and in marrow that expressed a PML/RARA transgene. Interestingly, although MYC and TRIB1 readily co-operated in leukemogenesis for wild-type bone marrow, TRIB1 provided no selective advantage to cells expressing PML/RARA. We hypothesized that this lack of co-operation between PML/RARA and TRIB1 reflected a common pathway for their effect: both proteins targeting the myeloid transcription factor C/EBPα. In support of this idea, TRIB1 expression abrogated the all-trans retinoic acid response of acute promyelocytic leukemia cells in vitro and in vivo Our data delineate the common and redundant inhibitory effects of TRIB1 and PML/RARA on C/EBPα providing a potential explanation for the lack of selection of TRIB1 in human acute promyelocytic leukemia, and highlighting the key role of C/EBPs in acute promyelocytic leukemia pathogenesis and therapeutic response. In addition, the co-operativity we observed between MYC and TRIB1 in the absence of PML/RARA show that, outside of acute promyelocytic leukemia, gain of both genes may drive selection for trisomy 8.

TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways.

The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.

Increased Alternative Macrophage-2 (M2) Polarization with Trib1 Gene over Expression in Myeloma Patients with Progressive Disease and Jak2 Inhibitors Reduce M2 Polarization

Introduction: The bone marrow (BM) microenvironment plays an important role in multiple myeloma (MM). The BM niche is composed of multiple cell types including macrophages. Macrophages polarize into pro-inflammatory macrophage-1 (M1) or alternative M2 states that promote tumor growth and metastasis. We evaluated the proportion of M2 macrophages in BM from MM pts either showing complete response (CR) or progressive disease (PD), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in macrophages, we also evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM.Methods: Using immunofluorescence (IFC), we determined the proportion of M1 and M2 macrophages in BM biopsies and aspirates from MM pts with PD or CR. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells. MM BM aspirates were also examined using flow cytometric analysis (FCA). Human monocytes isolated from healthy subjects or the THP1 monocyte cell line were co-cultured with MM cell lines (RPMI8226 and U266) or primary MM tumor cells. The effects of RUX at low concentrations (IC20) on M2 polarization were determined. The percentages of M1 and M2 macrophages were determined using FCA. Total RNA was extracted from monocytes. Quantitative PCR was measured with TaqMan technology. For the in vivo studies, human MM tumors (LAGκ-2) were surgically implanted into the left superficial gluteal muscle of SCID mice and tumor volume measured on a weekly basis.Results: The proportion of M2 macrophages (CD36+/ARG1+) was markedly increased in BM biopsies or mononuclear cells from MM pts with PD compared with those in CR using IFC staining. FCA also showed the percentage of M2 macrophages in BM was significantly increased in MM pts with PD (n=25) compared to those in CR (n=10; P=0.005) whereas there was no difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM pts with PD compared to those in CR. Trib1 gene mRNA levels were higher among pts with PD compared to those in CR whereas the gene expression of Trib2 and Trib3 was not different. Next, we co-cultured MM cell lines (U266) or fresh MM BMMCs with purified healthy human monocytes for one week. The percentage of M2 cells markedly increased and the proportion of M1 cells decreased. Trib1 gene expression increased during co-culture whereas there was no change in expression of the other two Tribs. When direct cell-to-cell contact occurred between the MM tumor cells and the monocytes, the percentage of M2 macrophages markedly increased. We investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. After exposure to a low concentration of RUX, the percentage of M2 cells decreased when the monocytes were co-cultured with MM tumor cells. Trib1 gene expression of the monocytes treated with RUX was also notably reduced compared with cells not treated with the JAK2 inhibitor. Using our human MM xenograft model LAGκ-2, RUX (1.5mg/kg) reduced tumor growth and decreased the proportion of M2 macrophages in the tumor tissue of MM tumor-bearing SCID mice.Conclusion: M2 cells are present at high levels in BM derived from MM pts with PD compared to those in CR, MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression. This induces monocyte differentiation into M2 macrophages that support MM tumor cell growth.. Notably, the JAK2 inhibitor RUX inhibits both M2 macrophage polarization and Trib1 gene expression in MM, and reduces tumor growth in SCID mice bearing human MM. These results suggest that RUX may be effective for treating MM pts. DisclosuresNo relevant conflicts of interest to declare.

Tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα.

Variants near the gene TRIB1 are significantly associated with several plasma lipid traits, circulating liver enzymes, and the development of coronary artery disease in humans; however, it is not clear how its protein product tribbles-1 regulates lipid metabolism. Here, we evaluated mice harboring a liver-specific deletion of Trib1 (Trib1_LSKO) to elucidate the role of tribbles-1 in mammalian hepatic lipid metabolism. These mice exhibited increased hepatic triglyceride (TG) content, lipogenic gene transcription, and de novo lipogenesis. Microarray analysis revealed altered transcription of genes that are downstream of the transcription factor C/EBPα, and Trib1_LSKO mice had increased hepatic C/EBPα protein. Hepatic overexpression of C/EBPα in WT mice phenocopied Trib1_LSKO livers, and hepatic knockout of Cebpa in Trib1_LSKO mice revealed that C/EBPα is required for the increased lipogenesis. Using ChIP-Seq, we found that Trib1_LSKO mice had increased DNA-bound C/EBPα near lipogenic genes and the Trib1 gene, which itself was transcriptionally upregulated by C/EBPα overexpression. Together, our results reveal that tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα, which in turn transcriptionally upregulates Trib1. These data suggest an important role for C/EBPα in mediating the lipogenic effects of hepatic Trib1 deletion and provide insight into the association between TRIB1 and plasma lipids, and liver traits in humans.

Association study of genetic variants at newly identified lipid gene TRIB1 with coronary heart disease in Chinese Han population.

BackgroundRecent genome-wide association studies (GWAS) have identified the variants near TRIB1 gene affecting blood lipid levels. However, the association between the reported variants and risk of coronary heart disease (CHD) was not confirmed.MethodsWe conducted two independent case–control studies. The first study consisted of 300 CHD patients and 300 controls and the second study had 1,332 CHD patients and 2,811 controls. The genotypes of two variants rs3201475 and rs17321515 in TRIB1 were determined by TaqMan assay. The dual-luciferase reporter assay was performed for evaluating the function of the SNP rs3201475.ResultsThe statistical analysis indicated that single nucleotide polymorphism (SNP) rs17321515 was replicated to be associated with triglyceride (TG) level, which was also significantly associated with CHD risk when using the stratified analysis after adjusting for conventional risk factors. Compared with GG genotype, AA carriers of SNP rs17321515 had higher risk in males (odds ratio (OR) = 1.28, 95 %CI = 1.01–1.61; P = 0.03) and smokers (OR = 1.41, 95%CI = 1.09–1.88; P = 0.01). We did not find significantly association between genotypes of rs3201475 and CHD risk. In addition, no significant difference was found in the luciferase activity assay of SNP rs3201475.ConclusionsOur findings indicated that SNP rs17321515 is significantly associated with plasma TG level and the increasing risk of CHD among males and smokers in Chinese, whereas there is no positive association between SNP rs3201475 and CHD risk. Smoking could modify the effects of TRIB1 on CHD risk.

Correlation between TRIB3 gene rs2295490 polymorphism and coronary heart disease with dyslipidemia

Objective To explore the correlation between the polymorphism of TRIB3 gene rs2295490 and coronary heart disease(CHD) with dyslipidemia. Methods In the case control study, we enrolled 220 CHD patients as observation group (60.15 years mean age, 150 males and 70 females) and 151 age and gender matched healthy individuals as control group (59.22 years mean age, 102 males and 49 fe males). Genotypes of TRIB3 gene rs2295490 polymorphisms in the two groups were detected by PCR and sequence-based testing.At the same time, blood glucose(GLU), total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol(HDL-C), low density lipoprotein cholesterol(LDL-C) were measured.The correlations between the polymorphism of TRIB3 gene rs2295490 and CHD, biochemical indicators of two groups were analyzed, respectively.The comparison of genotype and allele frequency between two groups used χ2 test, while the comparison of biochemical indexes between two groups used t test. Results In the control group, genotypic frequencies of AA, AG and GG of TRIB3 gene rs2295490 were 57%, 35% and 8%, and the frequencies of A and G alleles were 75% and 25%, respectively.In the CHD group, genotypic frequencies of AA, AG and GG of TRIB3 gene rs2295490 were 50%, 41% and 9%, and the frequencies of A and G alleles were 71% and 39%, respectively.There was no significant difference of genotype and allele frequencies of polymorphism of TRIB3 gene rs2295490 between CHD and healthy controls(χ2=1.74, P=0.42; χ2=1.26, P=0.243). In the CHD group, the HDL-C levels of the AA genotype were higher than those of AG+ GG genotype(t=-9.78, P=0.00); The TG, LDL-C levels of the AA genotype were lower than those of AG+ GG genotype(t=2.59, P=0.01; t=6.15, P=0.00). All the differences were statistically significant. Conclusions TRIB3 gene rs2295490 polymorphism has no correlation with CHD, but the G allele may be correlated with dyslipidemia in CHD patients .(Chin J Lab Med, 2015, 38: 273-276) Key words: Coronary disease; Dyslipidemias; Repressor proteins; Protein-serine-threonine kinases; Cell cycle proteins; Polymorphism, genetic

Functional variants of lipid level modifier MLXIPL, GCKR, GALNT2, CILP2, ANGPTL3 and TRIB1 genes in healthy Roma and Hungarian populations.

The role of triglyceride metabolism in different diseases, such as cardiovascular or cerebrovascular diseases is still under extensive investigations. In genome-wide studies several polymorphisms have been reported, which are highly associated with plasma lipid level changes. Our goal was to examine eight variants: rs12130333 at the ANGPTL3, rs16996148 at the CILP2, rs17321515 at the TRIB1, rs17145738 and rs3812316 of the MLXIPL, rs4846914 at GALNT2, rs1260326 and rs780094 residing at the GCKR loci. A total of 399 Roma (Gypsy) and 404 Hungarian population samples were genotyped using PCR-RFLP method. Significant differences were found between Roma and Hungarian population samples in both MLXIPL variants (C allele frequency of rs17145738: 94.1% vs. 85.6%, C allele frequency of rs3812316: 94.2% vs. 86.8% in Romas vs. in Hungarians, p < 0.05), in ANGPTL3 (T allele frequency of rs1213033: 12.2% vs. 18.5% in Romas vs. Hungarians, p < 0.05) and GALNT2 (G allele frequency of rs4846914: 46.6% vs. 54.5% Romas vs. in Hungarians, p < 0.05), while no differences over SNPs could be verified and the known minor alleles showed no correlation with triglyceride levels in any population samples. The current study revealed fundamental differences of known triglyceride modifying SNPs in Roma population. Failure of finding evidence for affected triglyceride metabolism shows that these susceptibility genes are much less effective compared for example to the apolipoprotein A5 gene.

Increased M2 Macrophages in Multiple Myeloma Patients with Progressive Disease and Down-Regulated Polarization with the JAK2 Inhibitor Ruxolitinib

Introduction: Macrophages polarize into pro-inflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. M2 cells promote tumor growth and metastasis through secretion of growth factors. However, crosstalk between tumor cells and macrophages in the development of M2 polarization has not been well demonstrated. We evaluated the proportion of M2 macrophages in bone marrow (BM) from patients (pts) with multiple myeloma (MM), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in the cell growth and differentiation of macrophages, we evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM.Methods: Using immunofluorescence (IFC) analysis, we determined the proportion of M1 and M2 macrophages in BM biopsies from MM pts with progressive disease and in remission. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells, following a standard IFC protocol. Monocyte/macrophage phenotypes for M1 and M2 subtypes in mononuclear cells isolated from MM BM aspirates were also examined using flow cytometric analysis with these same antibodies. Human monocytes isolated from normal subjects or the THP1 monocyte cell line were co-cultured with MM cell lines or primary MM tumor cells with or without exposure to low concentrations (IC20) of ruxolitinib (RUX) using Transwell plates. The percentage of M1 and M2 macrophages was determined using flow cytometric analysis. Total RNA was extracted from monocytes followed the manufacturer’s directions. Quantitation PCR were measured with TaqMan technology performed in an OneStepPlus instrument.Results: IFC demonstrated that the percentage of M2 macrophages (CD36+/ARG1+) was markedly increased in BM sections from MM pts with progressive disease compared with those pts in remission. The flow cytometric data also showed the percentage of M2 (CD36+/ ARG1+) macrophages in BM was significantly increased in MM patients with progressive disease (n=20) compared to those in remission (n=9; P=0.005) whereas there was no significant difference in the percentage of M1 (CD86+/iNOS+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. The results of both RT-PCR and Quantitation PCR showed Trib1 gene expression levels were higher among patients with progressive disease compared to those in remission. In contrast, the gene expression of Trib2 and Trib3 was not related to the MM patient’s clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured MM cell lines or fresh MM tumor cells with purified healthy human monocytes using Transwell plates. The percentage of M2 cells markedly increased whereas the proportion of M1 cells decreased. The expression of Trib1 increased during the 7 days of co-culture whereas there was no change in the expression of Trib2 and Trib3. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages markedly increased after 7 days of incubation. It has been reported that Trib-1 mediated regulation of the MAPK/ERK pathway in a murine model and the Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT3 whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Thus, we investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. The percentage of M2 cells was decreased when the monocytes that were co-cultured with MM tumor cells were treated with a low concentration (IC20) of RUX. Trib1 gene expression of the monocytes treated with RUX was also reduced comparing to the cells untreated with JAK2 inhibitor.Conclusion: Our results show that MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. M2 cells are present at high levels in BM derived from MM pts with progressive disease compared to those in remission. Notably, the JAK2 inhibitor RUX shows inhibition of both M2 macrophage polarization and Trib1 gene expression in MM, and these results suggest this drug may be effective for treating MM. DisclosuresNo relevant conflicts of interest to declare.

The functional Q84R polymorphism of TRIB3 gene is associated with diabetic nephropathy in Chinese type 2 diabetic patients

Increased oxidative stress and circulating free fatty acids (FFA) has been suggested to involve in the pathogenesis of diabetic nephropathy. TRIB3 can inhibit FFA and reactive oxygen species (ROS) stimulated podocyte production of MCP-1. Smoking increases the production of reactive oxygen species, which accelerates oxidative stress under hyperglycemia. To determine whether the Q84R polymorphism (rs2295490), alone or in combination with smoking, contributes to the development of diabetic nephropathy, a case–control study was performed in 812 Chinese patients with type 2 diabetes. Among patients, 214 had diabetic nephropathy with microalbuminuria (n=156) or overt albuminuria (n=58), and 598 did not show either of these symptoms but had diabetes for ≥10years and were not undergoing antihypertension treatment. After adjustment for confounders, TRIB3 single-nucleotide polymorphism rs2295490 was associated with DN (OR 1.318, 95% CI 1.075, 1.653, p=0.017); smoking was also an independent risk factor for diabetic nephropathy (1.42 [1.25–2.04], p<0.001). In addition, we identified possible synergistic effects; i.e., the high-risk group (smokers with the AG+GG genotype) showed 2.13 times higher risk (1.51–3.96, p<0.001) of diabetic nephropathy than the low-risk group (nonsmokers with the AA genotype) in a multiple logistic regression analysis controlled for the confounders, but no departure from additivity was found. Our results indicate that smoking and the TRIB3 G-allele is associated with an increased risk of diabetic nephropathy, which supports the hypothesis that oxidative stress contributes to the development of diabetic nephropathy.

Functional analysis of the TRIB1 associated locus linked to plasma triglycerides and coronary artery disease.

BackgroundThe TRIB1 locus has been linked to hepatic triglyceride metabolism in mice and to plasma triglycerides and coronary artery disease in humans. The lipid‐associated single nucleotide polymorphisms (SNPs), identified by genome‐wide association studies, are located ≈30 kb downstream from TRIB1, suggesting complex regulatory effects on genes or pathways relevant to hepatic triglyceride metabolism. The goal of this study was to investigate the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits.Methods and ResultsCharacterization of the risk locus reveals that it encompasses a gene, TRIB1‐associated locus (TRIBAL), composed of a well‐conserved promoter region and an alternatively spliced transcript. Bioinformatic analysis and resequencing identified a single SNP, rs2001844, within the promoter region that associates with increased plasma triglycerides and reduced high‐density lipoprotein cholesterol and coronary artery disease risk. Further, correction for triglycerides as a covariate indicated that the genome‐wide association studies association is largely dependent on triglycerides. In addition, we show that rs2001844 is an expression trait locus (eQTL) for TRIB1 expression in blood and alters TRIBAL promoter activity in a reporter assay model. The TRIBAL transcript has features typical of long noncoding RNAs, including poor sequence conservation. Modulation of TRIBAL expression had limited impact on either TRIB1 or lipid regulatory genes mRNA levels in human hepatocyte models. In contrast, TRIB1 knockdown markedly increased TRIBAL expression in HepG2 cells and primary human hepatocytes.ConclusionsThese studies demonstrate an interplay between a novel locus, TRIBAL, and TRIB1. TRIBAL is located in the genome‐wide association studies identified risk locus, responds to altered expression of TRIB1, harbors a risk SNP that is an eQTL for TRIB1 expression, and associates with plasma triglyceride concentrations.

Roles of TRIB3 in unfolded protein response

Endoplasmic reticulum stress in tumor cells is common,the accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can lead to unfolded protein response (UPR).Research shows that UPR can protect cells,reestablish endoplasmic reticulum function and homeostasis at initial stage,and can promote apoptotic at later stage.TRIB3 is one of the mammal pseudoprotein kinase family Tribbles,and plays an important pivotal role in UPR,which can not only promote apoptosis but also play a protective role. Key words: Neoplasms; Endoplasmic reticulum; TRIB3; Unfolded protein response

Reprogramming of hepatic lipoprotein metabolism by small molecule inducers of TRIB1 expression (998.1)

Recent genome wide association studies have identified TRIB1 as a novel locus associated with the risk of CAD and myocardial infarction. Since increased expression of the TRIB1 gene is associated with lower plasma levels of LDL cholesterol and triglycerides and higher levels of HDL cholesterol, we set up a qRT‐PCR‐based phenotypic assay for inducers of TRIB1 expression in human HepG2 cells. In the screen of the diversity‐oriented synthesis (DOS) compound collection we identified a class of benzofuran‐based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 overexpression as they inhibit the triglyceride synthesis and ApoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, encoding key proteins involved in lipoprotein assembly. Unexpectedly, active benzofurans ‐ as well as natural product TRIB1 inducers ‐ also modulate multiple components of hepatic cell cholesterol metabolism including elevating the expression of LDLR transcript and the level of LDL receptor while reducing the levels of PCSK9 transcript and secreted PCSK9 and stimulating the LDL uptake. The effects of the TRIB1 inducers are not masked by cholesterol depletion and are MAPKerk dependent. These chemically tractable compounds represent a novel type of modulators that reprogram cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging.

A case-control study indicates that the TRIB1 gene is associated with pancreatic cancer.

Pancreatic cancer is a malignant neoplasm originating from transformed cells arising in tissues that form the pancreas. To investigate whether the tribbles homolog 1 (Drosophila) gene (TRIB1) is associated with pancreatic cancer in the Chinese Han population, we conducted this case-control study and genotyped 3 single nucleotide polymorphisms (rs2980879, rs2980874, and rs2235108) of the TRIB1 gene in 182 patients and 359 normal controls of Chinese Han origin and analyzed their association. The results showed that the rs2980879 polymorphism was associated with pancreatic cancer [allele: P = 0.023434, genotype: P = 0.03005; odds ratio (OR) and 95% confidence interval (CI) = 0.727788 (0.552664-0.958404)], whereas the rs2980874 polymorphism had no association with pancreatic cancer [allele: P = 0.749885, genotype: P = 0.699533; OR and 95%CI = 1.041981 (0.809196-1.341734)], and the rs2235108 polymorphism was not associated with the disease [allele: P = 0.629475, genotype: P = 0.547534, OR and 95%CI = 1.128290 (0.690829-1.842770)]. Haplotype analyses and linkage disequilibrium tests were also conducted, and the results showed that these 3 loci are not in the same block. In conclusion, our study indicated that the TRIB1 gene is associated with pancreatic cancer. More studies with larger samples are needed in order to support this finding.

Protection from renal fibrosis, putative role of TRIB3 gene silencing

BackgroundRenal fibrosis is thought to be the common pathway in most cases of chronic kidney disease. Recently, TRIB3 was found to play an important role in progression of cardiac fibrosis in an insulin-resistant state. We investigated whether TRIB3 might participate in the pathogenesis of renal fibrosis in insulin-resistant rats. MethodsWe randomly separated 40 male Sprague–Dawley into 4 groups for treatment (n=10 each): control and high-fat diet (HFD) with TRIB3 siRNA adenovirus transfection, vehicle transfection or HFD alone. Insulin resistance markers were measured. Renal tissues were stained with hematoxylin and eosin, Masson's trichrome and periodic acid-Schiff. ResultsRats with HFD showed insulin resistance and TRIB3 overexpression. Upregulated TRIB3 expression could induce renal fibrosis accompanied by increased phosphorylation of extracellular signal-regulated kinase (ERK). Also, TRIB3 siRNA knockdown could ameliorate renal fibrosis, which was accompanied by decreased phosphorylation of ERK. ConclusionsTRIB3 gene silencing can attenuate renal fibrosis for beneficial effect on the development of renal fibrosis in chronic kidney disease in rat.

Increased M2 Macrophages and Higher Levels Of The Tribble Family Member Trib1 In Monocytes Are Associated With Progressive Disease In Multiple Myeloma (MM) Patients

Macrophages consist of two subgroups, M1 and M2. M1 macrophages are pro-inflammatory cells against bacterial and viral infections whereas M2 macrophages are anti-inflammatory and associated with tumor progression. The mammalian tribble (Trib) family of genes, Trib1, Trib2 and Trib3, encode pseudokinase proteins that have important roles in monocyte/macrophage proliferation, differentiation, and apoptosis. Trib1 is a critical factor that induces M2 macrophage differentiation in the bone marrow (BM).First, we investigated the proportion of M1 and M2 macrophages in BM mononuclear cells (MCs) from multiple myeloma (MM) patients with progressive disease or in remission using flow cytometric analysis. The percentage of M2 (CD36+/CD86-) macrophages in BM was significantly increased in MM patients with progressive disease (n=15) compared to those in remission (n=5; P<0.05) whereas there was no difference in the percentage of M1 (CD86+/CD14+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. Using immunohistochemical (IHC) analysis, the proportion of M2 macrophages was also determined in MM BM biopsies from patients with progressive disease and remission. The samples were cut into five-micrometer sections and double stained with two antibodies following a standard IHC protocol. IHC demonstrated that the percentage of M2 macrophages (CD36+/CD14+) was markedly increased in BM sections from MM patients with progressive disease compared to those in remission. In contrast, the percentage of M1 (CD86+/CD14+) macrophages was not different among those patients with progressive disease compared to those in remission. Next, we analyzed Trib1, Trib2 and Trib3 gene expression in BMMCs obtained from MM patients with progressive disease or in remission. RT-PCR results showed Trib1 expression levels were much higher among patients with progressive disease compared to those in remission. In contrast, the expression Trib2 and Trib3 was not related to the MM patient's clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured fresh MM tumor cells with purified healthy human monocytes. BMMCs from MM patients were co-cultured with human monocytes from normal subjects using Transwell plates and the percentage of M1 and M2 macrophages was determined using flow cytometric analysis following 2, 5 and 7 days of culture. The percentage of M2 cells increased whereas the proportion of M1 cells decreased. Gene expression of Trib1, Trib2 and Trib3 was analyzed using RT-PCR following 2, 5, and 7 days of co-culture. The expression of Trib 1 increased during the 7 days of co-culture whereas the expression of Trib2 and Trib3 did not change. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages (CD36+) markedly increased after 7 days of incubation.We have shown that MM cells induce monocytes to increase Trib1 gene expression, which stimulates M2 differentiation in monocytes. M2 cells, in turn, induce tumor progression, providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. Overall, we propose that Trib1 may be considered as a potential novel therapeutic target for the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

Elastography-based analysis of variants identified by a genome-wide association study as genetic determinants of liver enzyme activities

Background: Recent genome-wide study (GWAS) in over 61,000 individuals identified a set of loci that determine serum activities of liver enzymes [1]. Interestingly, only two variants, which are localized in the C6orf142 and TRIB1 gene loci, simultaneously increased the serum activities of ALT, AST and GGT [1]. Here we investigate these C6orf142 and TRIB1 variants as potential risk factors for increased liver stiffness in patients with chronic liver diseases (CLD).

Population structure of Hispanics in the United States: the multi-ethnic study of atherosclerosis.

Using ∼60,000 SNPs selected for minimal linkage disequilibrium, we perform population structure analysis of 1,374 unrelated Hispanic individuals from the Multi-Ethnic Study of Atherosclerosis (MESA), with self-identification corresponding to Central America (n = 93), Cuba (n = 50), the Dominican Republic (n = 203), Mexico (n = 708), Puerto Rico (n = 192), and South America (n = 111). By projection of principal components (PCs) of ancestry to samples from the HapMap phase III and the Human Genome Diversity Panel (HGDP), we show the first two PCs quantify the Caucasian, African, and Native American origins, while the third and fourth PCs bring out an axis that aligns with known South-to-North geographic location of HGDP Native American samples and further separates MESA Mexican versus Central/South American samples along the same axis. Using k-means clustering computed from the first four PCs, we define four subgroups of the MESA Hispanic cohort that show close agreement with self-identification, labeling the clusters as primarily Dominican/Cuban, Mexican, Central/South American, and Puerto Rican. To demonstrate our recommendations for genetic analysis in the MESA Hispanic cohort, we present pooled and stratified association analysis of triglycerides for selected SNPs in the LPL and TRIB1 gene regions, previously reported in GWAS of triglycerides in Caucasians but as yet unconfirmed in Hispanic populations. We report statistically significant evidence for genetic association in both genes, and we further demonstrate the importance of considering population substructure and genetic heterogeneity in genetic association studies performed in the United States Hispanic population.

TRIB1 constitutes a molecular link between regulation of sleep and lipid metabolism in humans

Epidemiological studies show association between sleep duration and lipid metabolism. In addition, inactivation of circadian genes induces insulin resistance and hyperlipidemia. We hypothesized that sleep length and lipid metabolism are partially controlled by the same genes. We studied the association of total sleep time (TST) with 60 genetic variants that had previously been associated with lipids. The analyses were performed in a Finnish population-based sample (N=6334) and replicated in 2189 twins. Finally, RNA expression from mononuclear leucocytes was measured in 10 healthy volunteers before and after sleep restriction. The genetic analysis identified two variants near TRIB1 gene that independently contributed to both blood lipid levels and to TST (rs17321515, P=8.92*10−5, Bonferroni corrected P=0.0053, β=0.081 h per allele; rs2954029, P=0.00025, corrected P=0.015, β=0.076; P<0.001 for both variants after adjusting for blood lipid levels or body mass index). The finding was replicated in the twin sample (rs17321515, P=0.022, β=0.063; meta-analysis of both samples P=8.1*10−6, β=0.073). After the experimentally induced sleep restriction period TRIB1 expression increased 1.6-fold and decreased in recovery phase (P=0.006). In addition, a negative correlation between TRIB1 expression and slow wave sleep was observed in recovery from sleep restriction. These results show that allelic variants of TRIB1 are independently involved in regulation of lipid metabolism and sleep. The findings give evidence for the pleiotropic nature of TRIB1 and may reflect the shared roots of sleep and metabolism. The shared genetic background may at least partially explain the mechanism behind the well-established connection between diseases with disrupted metabolism and sleep.

Role of TRIB3 in Diabetic and Overnutrition-Induced Atherosclerosis

Obesity caused by excess feeding (overnutrition) has become a problem of epidemic proportions and is the underlying cause in metabolic disorders and chronic diseases such as diabetes and cardiovascular disease. Overnutrition is associated with systemic and tissue-related insulin resistance, an abnormality that promotes vascular disease as well as the development of diabetes (1,2). Thus, there is considerable interest in factors that link overnutrition, insulin resistance, and hyperglycemia with vascular disease. There is emerging evidence that the expression of the Tribbles homolog 3 of Drosophila ( TRIB3 ) gene is increased in patients and animals with type 2 diabetes (3). The TRIB3 gene is located on the 20p13 region of the human chromosome. Its full-length translated mRNA is 1,074 base pairs, and its protein product is made up of 358 amino acids. Studies have shown that TRIB3 inhibits insulin metabolic signaling in liver (4–6), skeletal muscle (7), and vascular tissue (8). Further, these studies suggest that TRIB3 expression in skeletal muscle and liver tissue is increased with excessive nutrient intake as well as by hyperglycemia (3–7). Endoplasmic reticulum stress has also been shown to increase TRIB3 gene expression, and TRIB3 promotes cell death in response to endoplasmic reticulum stress (9) (Fig. 1). Several studies have shown that TRIB3 impairs insulin …