Amy Martinuk

Functional Analysis of the TRIB1 Associated Locus Linked to Plasma Triglycerides and Coronary Artery Disease

Background The 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 Results Characterization 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. Conclusions These 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.

Characterization of a long-acting recombinant human serum albumin-atrial natriuretic factor (ANF) expressed in Pichia pastoris

The cardiac hormone atrial natriuretic factor (ANF) combines pharmacological properties of drugs used to treat essential hypertension (EH), congestive heart failure (CHF) and acute myocardial infarction (AMI). Treatment of CHF or AMI patients with an intravenous (iv) infusion of the circulating form of ANF (ANF(99-126)) produces significant clinical improvement. The short half-life (5 min) and peptide nature of ANF impose logistic restrictions for chronic administration. To increase its half-life, we fused ANF and human serum albumin (HSA) mini-genes by recombination in Pichia pastoris. The activity of three configurations of the fusion protein was tested in vitro and in vivo. The fusion protein that comprised of C-terminus HSA connected to N-terminus ANF via a hexaglycine linker showed the best outcome; it increased cGMP production in vitro. In vivo an iv bolus of HSA-ANF into mice increased significantly plasma cGMP levels and lowered blood pressure (BP) for up to 6 h hence successfully extended ANF half-life in plasma while retaining its biological activity. HSA-ANF represents the basis for development in the chronic therapeutic use of ANF.

Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Objective—A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-&bgr; pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease–associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. Approach and Results—By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D′, 0.97; r2, 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. Conclusions—The coronary artery disease–associated rs17293632C>T single nucleotide polymorphism represents a novel functional cis-acting element at the SMAD3 locus. The protective (T) allele of rs17293632 disrupts a consensus AP-1 binding site in a SMAD3 intron 1 enhancer, reduces enhancer activity and SMAD3 expression, altering human arterial smooth muscle cell proliferation.

Cardiac hormones ANF and BNP modulate proliferation in the unidirectional mixed lymphocyte reaction

BACKGROUND Previous investigations have shown that the plasma levels of the cardiac hormone brain natriuretic peptide (BNP) increase during acute cardiac allograft rejection as diagnosed by endomyocardial biopsy. Successful immunosuppressant treatment decreased plasma BNP levels, suggesting a role for BNP in transplantation immunity. We tested a possible immunomodulatory effect of the natriuretic peptides (NPs) BNP, atrial natriuretic factor (ANF), and C-type NP (CNP) using the unidirectional mixed lymphocyte reaction (MLR). METHODS Lymphocytes were isolated from the lymph nodes of Brown Norway (BN) and Lewis (L) rats. BN lymphocytes were gamma-irradiated to inhibit DNA synthesis. Lymphocytes at 2.5 x 10(6) cell/ml were mixed (at an L:BN ratio of 4:1) and incubated. On Days 2 and 3, ANF (10(-6) to 10(-11) mol/liter), BNP (10(-5) to 10(-11) mol/liter), or CNP (10(-6) to 10(-12) mol/liter) were added. Cell proliferation was measured on Day 4. RESULTS Reverse transcript-polymerase chain reaction (RT-PCR) analysis of BN and L lymphocytes detected NP receptor (NPR) mRNA amplicons of the expected size. MLR induced an increase in relative receptor abundance as follows: NPRA > NPRB > NPRC. ANF and BNP significantly inhibited up to approximately 50% lymphocyte proliferation in a dose-dependent manner in the range of 10(-11) to 10(-6) mol/liter, whereas CNP significantly decreased lymphocyte proliferation only modestly (approximately 20%) at 10(-8) mol/liter and at 10(-6) mol/liter. CONCLUSIONS Both ANF and BNP have immunomodulatory functions, although the response to cardiac rejection observed clinically involves increases in plasma levels of BNP only. This is likely related to BNP gene promoter sequences previously reported to be responsive to specific cytokines and related substances. The modulation of the MLR by NP suggests a possible clinical use of these peptides in transplantation immunity.

Functional interaction between COL4A1/COL4A2 and SMAD3 risk loci for coronary artery disease

OBJECTIVE The COL4A1/COL4A2 region on chromosome 13q34 is a highly replicated locus for coronary artery disease (CAD). In the normal arterial wall, type IV collagen acts to inhibit smooth muscle cell proliferation. Its production is in part a function of TGFβ signaling, but the specific regulatory mechanisms, especially in humans, have not been defined. Our aim was to decipher TGFβ signaling components important in the regulation of COL4A1 and COL4A2 and determine whether these components showed genetic interaction with the COL4A1/COL4A2 locus for CAD association. METHODS AND RESULTS Experiments were performed in primary human aortic smooth muscle cells and HT1080 fibroblasts. Pharmacological inhibition of the TGFβ1 receptor and subsequent SMAD protein phosphorylation by treatment with an ALK5 inhibitor prevented the increase in COL4A1/COL4A2 mRNA (p < 0.001) and protein expression in response to TGFβ1 stimulation. In contrast, inhibition of the non-canonical TGFβ signaling pathways was without effect. siRNA mediated knockdown of SMAD3 and SMAD4 abolished the stimulatory effects of TGFβ1 on COL4A1/COL4A2 (p < 0.001) whereas SMAD2 knockdown had no effect. In luciferase reporter assays, neither SMAD3 overexpression nor TGFβ1 treatment altered COL4A1 or COL4A2 promoter activity, supportive of more complex regulation of type IV collagen gene expression by the TGFβ/SMAD3 signaling pathway. Epistasis analysis in 5 CAD case/control cohorts revealed that SMAD3 and COL4A1/COL4A2 display statistical interaction for CAD association. CONCLUSIONS These findings demonstrate that SMAD3 is a necessary factor for TGFβ-mediated stimulation of mRNA and protein expression of type IV collagen genes in human vascular smooth muscle cells. Epistasis analyses further supports the hypothesis that the SMAD3-dependent regulation of COL4A1/COL4A2 may be of functional significance for CAD pathogenesis.

ERK1/2 regulates hepatocyte Trib1 in response to mitochondrial dysfunction.

The TRIB1 locus (8q24.13) is a novel locus identified and replicated by several genome-wide association studies for associations with plasma triglycerides, apolipoprotein B and coronary artery disease. The TRIB1 protein product, tribbles-like protein 1 (Trib1), regulates MAPK activity. MAP kinases transduce a large variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation and apoptosis. Importantly, Trib1 has been shown to regulate hepatic lipogenesis and very low density lipoprotein production. Despite the relevance of hepatocyte Trib1 to lipid metabolism and atherosclerosis, little is known about the mechanisms regulating Trib1 itself. Here, we identify the mitochondria axis as a regulator of Trib1. Treatment of HepG2 cells with a short pulse of a low oligomycin concentration led to a potent and prolonged increase in the Trib1 mRNA, an effect that was shared with other mitochondria stressors. HuH7 cells as well murine hepatocytes were also responsive albeit to a weaker extent. The upregulation appeared largely independent of reactive oxygen species generation or metabolic stress and was mainly under transcriptional control, with ERK1/2 playing an important regulating role in the process. While the presence of the Trib1 protein could be inferred, attempts to correlate the increased mRNA to changes in protein level were unsuccessful due to the lack of recognizable Trib1 signal. Our data enrich the current paradigm of Trib1 as an activator of the MAPK pathway by uncovering a role for MAPK in regulating Trib1.

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.

Role of Tribbles Pseudokinase 1 (TRIB1) in human hepatocyte metabolism

Genome-wide association studies for plasma triglycerides and hepatic steatosis identified a risk locus on chromosome 8q24 close to the TRIB1 gene, encoding Tribbles Pseudokinase 1 (TRIB1). In previous studies conducted in murine models, hepatic over-expression of Trib1 was shown to increase fatty acid oxidation and decrease triglyceride synthesis whereas Trib1 knockdown mice exhibited hypertriglyceridemia. Here we have examined the impact of TRIB1 suppression in human and mouse hepatocytes. Examination of a panel of lipid regulator transcripts revealed species-specific effects, prompting us to focus on human models for the remainder of the study. Acute knockdown of TRIB1 in human primary hepatocytes resulted in decreased expression of MTTP and APOB, required for very low density lipoprotein (VLDL) assembly although particle secretion was not significantly affected. A parallel analysis performed in HepG2 revealed reduced MTTP, but not APOB, protein as a result of TRIB1 suppression. Global gene expression changes of human primary hepatocytes upon TRIB1 suppression were analyzed by clustering algorithms and found to be consistent with dysregulation of several pathways fundamental to liver function, including altered CEBPA and B transcript levels and impaired glucose handling. Indeed, TRIB1 expression in HepG2 cells was found to be inversely proportional to glucose concentration. Lastly TRIB1 downregulation in primary hepatocytes was associated with suppression of the HNF4A axis. In HepG2 cells, TRIB1 suppression resulted in reduced HNF4A protein levels while HNF4A suppression increased TRIB1 expression. Taken together these studies reveal an important role for TRIB1 in human hepatocyte biology.

TRIB1 is a positive regulator of hepatocyte nuclear factor 4-alpha

The TRIB1 locus has been linked to both cardiovascular disease and hepatic steatosis. Recent efforts have revealed TRIB1 to be a major regulator of liver function, largely, but not exclusively, via CEBPA degradation. We recently uncovered a functional interaction between TRIB1 and HNF4A, another key regulator of hepatic function, whose molecular underpinnings remained to be clarified. Here we have extended these findings. In hepatoma models, HNF4A levels were found to depend on TRIB1, independently of its impact on CEBPA. Using a reporter assay model, MTTP reporter activity, which depends on HNF4A, positively correlated with TRIB1 levels. Confocal microscopy demonstrated partial colocalization of TRIB1 and HNF4A. Using overexpressed proteins we demonstrate that TRIB1 and HNF4A can form complexes in vivo. Mapping of the interaction interfaces identified two distinct regions within TRIB1 which associated with the N-terminal region of HNF4A. Lastly, the TRIB1-HNF4A interaction resisted competition with a CEPBA-derived peptide, suggesting different binding modalities. Together these findings establish that TRIB1 is required for HNF4A function. This regulatory axis represents a novel CEBPA-independent aspect of TRIB1 function predicted to play an important role in liver physiology.

Functional Validation of a Common Nonsynonymous Coding Variant in ZC3HC1 Associated With Protection From Coronary Artery DiseaseCLINICAL PERSPECTIVE

Background— Although virtually all coronary artery disease associated single-nucleotide polymorphisms identified by genome-wide association studies (GWAS) are in noncoding regions of the genome, a common polymorphism in ZC3HC1 (rs11556924), resulting in an arginine (Arg) to histidine (His) substitution in its encoded protein, NIPA (Nuclear Interacting Partner of Anaplastic Lyphoma Kinase) is linked to a protection from coronary artery disease. NIPA plays a role in cell cycle progression, but the functional consequences of this polymorphism have not been established. Methods and Results— Here we demonstrate that total ZC3HC1 expression in whole blood is similar across genotypes, despite expression being slightly biased toward the risk allele in heterozygotes. At the protein level, the protective His363 NIPA variant exhibits increased phosphorylation of a critical serine residue (Ser354) and higher protein expression as compared with the Arg363 variant. Binding experiments indicate that neither SKP1 (S-phase kinase-associated protein 1) nor CCNB1 binding were affected by the polymorphism. Despite similar nuclear distribution, NIPA His363 exhibits greater nuclear mobility. NIPA suppression results in a modest reduction of proliferation in vascular smooth muscle cells, but given low proliferative capacity, a significant effect of the variant was not noted. By contrast, we demonstrate that the protective variant reduces cell proliferation in HeLa cells. Conclusions— These findings extend the genetic association between rs11556924 and coronary artery disease risk by characterizing its effects on the encoded protein, NIPA. The resulting amino acid change Arg363His is associated with increased expression and nuclear mobility, as well as lower rates of cell growth in HeLa cells, further supporting a role for cell proliferation in atherosclerosis and its clinical consequences.