👤 C Cheng

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)II has been shown to play a major role in endothelial cell growth and regulation of the Notch signaling pathway to confer vein identity. However, the underlying mechanisms for COUP-TFII regulation in these pathways remain to be defined. Here we employed a genomic approach by using microarray analysis to identify downstream targets in human umbilical vein endothelial cells (HUVEC) cells and found the expression of many genes in the cell cycle pathway and Notch signaling pathway are significantly altered in the COUP-TFII-depleted cells. The expression of E2F transcription factor 1 (E2F1), a key transcription factor that regulates the expression of cell cycle regulators, is reduced in the absence of COUP-TFII. Using chromatin immunoprecipitation experiments, we showed that COUP-TFII directly regulates the expression of E2F1 through tethering to the Sp1 binding sites in the promoter of E2F1 to modulate cell proliferation. In addition, we also demonstrate that Foxc1 and Np-1, two upstream genes of Notch signaling and Hey2, a downstream effector of Notch signaling, are direct targets of COUP-TFII. Furthermore, COUP-TFII suppresses the expression of EphrinB2, an arterial marker, while enhancing the expression of ephrin receptor B4, a venous marker, supporting our in vivo findings that COUP-TFII regulates vein identity by suppressing the Notch signal pathway. Show less

Lygodipenoids A (1) and B (2), two novel C33 tetracyclic triterpenoids with a new 9,19 : 24,32-dicyclopropane skeleton, were isolated from the whole grass of Lygodium japonicum. Their structures were elucidated by spectroscopic and chemical means. Compounds 1 and 2 were tested in transfected cultured human embryonic kidney 293 HEK293 cells for an agonist assay, and compound 1 was identified as a partial agonist for liver X receptor α. Show less

Selenium neutralizes interleukin-1β (IL-1β) induced inflammatory responses in chondrocytes. We investigated potential mechanisms for this through in vitro knock down of three major selenoproteins, Iodothyronine Deiodinase-2 (DIO2), Glutathione Peroxidase-1 (GPX1), and Thioredoxin Reductase-1 (TR1) in primary human chondrocytes. Primary human chondrocytes were transfected with scrambled small interfering ribonucleic acid (siRNA) or siRNA specific for DIO2, GPX1 and TR1. After 48 h, transfected cells were cultured in serum free media for 48 h, with or without 10 pg/ml IL-1β for the final 24h. The efficiency of siRNAs was confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot analysis. The gene expression, by qRT-PCR, of cyclooxygenase-2 (COX2), IL-1β, and Liver X receptor (LXR) alpha and beta was evaluated to determine the impact of selenoprotein knockdown on inflammatory responses in chondrocytes. The messenger RNA (mRNA) expression of DIO2, GPX1, and TR1 was significantly decreased by the specific siRNAs (reduced 56%, P=0.0004; 96%, P<0.0001; and 66%, P<0.0001, respectively). Suppression of DIO2, but not GPX1 or TR1, significantly increased (~2-fold) both basal (P=0.0005) and IL-1β induced (P<0.0001) COX2 gene expression. Similarly, suppression of DIO2 significantly increased (∼9-fold) IL-1β induced IL-1β gene expression (P=0.0056) and resulted in a 32% (P=0.0044) decrease in LXRα gene expression but no effect on LXRβ. Suppression of the selenoprotein DIO2 resulted in strong pro-inflammatory effects with increased expression of inflammatory mediators, IL-1β and COX2, and decreased expression of LXRα suggesting that this may be the upstream target through which the anti-inflammatory effects of DIO2 are mediated. Show less

Genome-wide association studies have identified common genetic variants that can contribute specifically to the risk of abdominal adiposity, as measured by waist circumference or waist-to-hip ratio. However, it is unknown whether these genetic risk factors affect relative body fat distribution in the abdominal visceral and subcutaneous compartments. The association between imaging-based abdominal fat mass and waist-size risk variants in the FTO, LEPR, LYPLAL1, MSRA, NRXN3, and TFAP2B genes was investigated. A cross-sectional sample of 60 women was selected among study participants of The Multiethnic Cohort, who were aged 60 to 65 years, of European or Japanese descent, and with a body mass index (calculated as kg/m(2)) between 18.5 and 40. Dual-energy x-ray absorptiometry and abdominal magnetic resonance imaging scans were used to measure adiposity. After adjustments for age, ethnicity, and total fat mass, the FTO variants showed an association with less abdominal subcutaneous fat and a higher visceral-to-subcutaneous abdominal fat ratio, with the variant rs9941349 showing significant associations most consistently (P=0.003 and 0.03, respectively). Similarly, the LEPR rs1137101 variant was associated with less subcutaneous fat (P=0.01) and a greater visceral-to-subcutaneous fat ratio (P=0.03) and percent liver fat (P=0.007). MSRA rs545854 variant carriers had a lower percent of leg fat. Our findings provide initial evidence that some of the genetic risk factors identified for larger waist size might also contribute to disproportionately greater intra-abdominal and liver fat distribution in postmenopausal women. If replicated, these genetic variants can be incorporated with other biomarkers to predict high-risk body fat distribution. Show less

Obesity is a leading contributor to colorectal cancer risk. We investigated whether the risk variants identified in genome-wide association studies of body mass index (BMI) and waist size are associated with colorectal cancer risk, independently of the effect of obesity phenotype due to a shared etiology. Twenty-four single nucleotide polymorphisms (SNPs) in 15 loci (BDNF, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MC4R, MSRA, MTCH2, NEGR1, NRXN3, SEC16B, SH2B1, TFAP2B and TMEM18) were genotyped in a case-control study of 2,033 colorectal cancer cases and 9,640 controls nested within the multiethnic cohort study, as part of the population architecture using genomics and epidemiology consortium. Risk alleles for two obesity SNPs were associated with colorectal cancer risk--KCTD15 rs29941 [odds ratio (OR) for C allele = 0.90, 95% confidence interval (CI) 0.83-0.98; p = 0.01] and MC4R rs17782313 (OR for C allele = 1.12, 95% CI 1.02-1.22; p = 0.02). These associations were independent of the effect of BMI. However, none of the results remained significant after adjustment for multiple comparisons. No heterogeneity was observed across race/ethnic groups. Our findings suggest that the obesity risk variants are not likely to affect the risk of colorectal cancer substantially. Show less

In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance. Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type immune receptors operates largely independently of both genes, demonstrating specific contributions to plant defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis. Show less

The objectives of this study were to synthesize and characterize two types of cytarabine (Ara-C) lipid produgs and evaluate the prodrugs for sustained intraocular delivery after administration by intravitreal injection. Hexadecyloxypropyl cytarabine 5'-monophosphate (HDP-P-Ara-C) and hexadecyloxypropyl cytarabine 3',5'-cyclic monophosphate (HDP-cP-Ara-C) were synthesized starting from cytarabine (1-β-D-arabinofuranosylcytosine). Their vitreal clearance profile was simulated using a custom dissolution chamber, in vitro cytotoxicity was evaluated using cell proliferation assays, and in vivo ocular properties in rat and rabbit eyes were assessed using biomicroscopy, indirect ophthalmoscopy, tonometry, electroretinography, and histology. HDP-P-Ara-C was cleared from the dissolution chamber (flow rate 2 µL/min) within 7 days. In contrast, HDP-cP-Ara-C, a much more insoluble prodrug, was still detectable 36 days after the dissolution process was started. HDP-P-Ara-C had a 50% cytotoxicity concentration of 52±2.6 μM in human retinal pigment epithelium (ARPE-19) and 32±2.2 µM in a rat Müller cell line, rMC-1. The 50% cytotoxicity concentration values for HDP-cP-Ara-C in ARPE-19 and rMC-1 cells were 50 µM and 25 µM, respectively. HDP-P-Ara-C was not detectable 2 weeks after the highest intravitreal dose (228 µg/rat eye) was injected, and no ocular toxicity was found. With HDP-cP-Ara-C, the drug depot was visible for 26 weeks following a single intravitreal injection (800 µg/rabbit eye). For both compounds, the electroretinogram, intraocular pressure, and other toxicity studies were negative except for the highest dose of HDP-cP-Ara-C (800 µg/eye), which had focal toxicity from the direct touch of the retina and decreased dark adapted a-waves and decreased flicker electroretinogram amplitudes (generalized estimating equations, p=0.039 and 0.01). The cyclic monophosphate prodrug, HDP-cP-Ara-C, was found to have physiochemical properties better suited for sustained delivery of cytarabine to posterior segments of the eye. These properties included limited aqueous solubility, in vitro antiproliferative activity, and good tolerability after injection into rabbit eyes. Show less

Although recent studies have shown that human genomes contain hundreds of loci that exhibit signatures of positive selection, variants that are associated with adaptation in energy-balance regulation remain elusive. We reasoned that the difficulty in identifying such variants could be due to heterogeneity in selection pressure and that an integrative approach that incorporated experiment-based evidence and population genetics-based statistical judgments would be needed to reveal important metabolic modifiers in humans. To identify common metabolic modifiers that underlie phenotypic variation in diabetes-associated or obesity-associated traits in humans, or both, we screened 207 candidate loci for regulatory single nucleotide polymorphisms (SNPs) that exhibited evidence of gene-environmental interactions. Three SNPs (rs3895874, rs3848460, and rs937301) at the 5' gene region of human GIP were identified as prime metabolic-modifier candidates at the enteroinsular axis. Functional studies have shown that GIP promoter reporters carrying derived alleles of these three SNPs (haplotype GIP(-1920A)) have significantly lower transcriptional activities than those with ancestral alleles at corresponding positions (haplotype GIP(-1920G)). Consistently, studies of pregnant women who have undergone a screening test for gestational diabetes have shown that patients with a homozygous GIP(-1920A/A) genotype have significantly lower serum concentrations of glucose-dependent insulinotropic polypeptide (GIP) than those carrying an ancestral GIP(-1920G) haplotype. After controlling for a GIPR variation, we showed that serum glucose concentrations of patients carrying GIP(-1920A/A) homozygotes are significantly higher than that of those carrying an ancestral GIP(-1920G) haplotype (odds ratio 3.53). Our proof-of-concept study indicates that common regulatory GIP variants impart a difference in GIP and glucose metabolism. The study also provides a rare example that identified the common variant-common phenotypic variation pattern based on evidence of moderate gene-environmental interactions. Show less

The innate immune system detects viral infection predominantly by sensing viral nucleic acids. We report the identification of a viral sensor, consisting of RNA helicases DDX1, DDX21, and DHX36, and the adaptor molecule TRIF, by isolation and sequencing of poly I:C-binding proteins in myeloid dendritic cells (mDCs). Knockdown of each helicase or TRIF by shRNA blocked the ability of mDCs to mount type I interferon (IFN) and cytokine responses to poly I:C, influenza A virus, and reovirus. Although DDX1 bound poly I:C via its Helicase A domain, DHX36 and DDX21 bound the TIR domain of TRIF via their HA2-DUF and PRK domains, respectively. This sensor was localized within the cytosol, independent of the endosomes. Thus, the DDX1-DDX21-DHX36 complex represents a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses in the cytosol of mDCs. Show less

Alternative splicing (AS) is a widespread mechanism underlying the generation of proteomic and regulatory complexity. However, which of the myriad of human AS events play important roles in disease is largely unknown. To identify frequently occurring AS events in lung cancer, we used AS microarray profiling and reverse transcription-PCR (RT-PCR) assays to survey patient-matched normal and adenocarcinoma tumor tissues from the lungs of 29 individuals diagnosed with non-small cell lung cancer (NSCLC). Of 5,183 profiled alternative exons, four displayed tumor-associated changes in the majority of the patients. These events affected transcripts from the VEGFA, MACF1, APP, and NUMB genes. Similar AS changes were detected in NUMB and APP transcripts in primary breast and colon tumors. Tumor-associated increases in NUMB exon 9 inclusion correlated with reduced levels of NUMB protein expression and activation of the Notch signaling pathway, an event that has been linked to tumorigenesis. Moreover, short hairpin RNA (shRNA) knockdown of NUMB followed by isoform-specific rescue revealed that expression of the exon 9-skipped (nontumor) isoform represses Notch target gene activation whereas expression of the exon 9-included (tumor) isoform lacks this activity and is capable of promoting cell proliferation. The results thus reveal widespread AS changes in NSCLC that impact cell signaling in a manner that likely contributes to tumorigenesis. Show less

To investigate the effect of Hepatitis B Virus X Protein (HBx) on the expression of lipid metabolism-related genes and its role in pathogenesis of hepatocyte fatty degeneration. Hepatitis B Virus X gene eukaryon expression vector pIRES2-eGFP-HBx was transfected into HepG2 cells to establish HepG2/HBx cell model for HBx expression. HepG2 cells transfected with pIRES2-eGFP (HepG2/pIRES2 cell) and non-transfected were used as controls. At 24, 48 and 72 hours after transfection, the expression of green fluorescent protein (GFP) was observed by fluorescence microscope and the triglyceride(TG) content was detected. RT-PCR and Western blot were applied to detect the levels of sterol regulatory element binding protein-1 (SREBP-1), liver x receptor alpha (LXRalpha) mRNA and the levels of HBx, LXRalpha and fatty acid synthase (FAS) protein. At 24, 48 and 72 hours after transfection, the expression of GFP was found in HepG2/HBx and HepG2/pIRES2 cells, and increased gradually. The expression of HBx was detected only in HepG2/HBx cells, and was increased with time after transfection (F = 32.21, P less than 0.01). These suggested successful obtaining of HepG2-HBx cell model for HBx expression. At 24h, 48h and 72h after transfection, the expression levels of LXRalpha mRNA (0.386+/-0.055, 0.505+/-0.071, 0.649+/-0.058 ) and SREBP-1 mRNA (0.395+/-0.055, 0.548+/-0.047, 0.795+/-0.058), as well as the levels of LXRalpha protein(0.178+/-0.036, 0.263+/-0.047, 0.347+/-0.058) and FAS protein(0.436+/-0.055, 0.608+/-0.053, 0.827+/-0.046) in HepG2-HBx group were dramatically higher than those in the controls at the same time points (all P less than 0.05/0.01), and were gradually increased with time (all P less than 0.05/0.01). A positive correlationship was observed between HBX protein level and the LXRalpha, SREbP-1 mRNA and LXRalpha, FAS protein levels. The difference of TG content between HepG2/HBx group and control groups was not statistically significant (P more than 0.05). HBx-LXRalpha-SREBP-1/FAS pathway suggested regulating transcription and expression of lipid metabolism-related genes, which might be one of the important molecular mechanism causing hepatocyte fatty degeneration. Show less

The present study was designed to investigate the myocardial expression of liver X receptor alpha (LXRα) in a streptozotocin (STZ)-induced diabetic rat model. Immunohistochemical staining, quantitative real-time RT-PCR, and Western blot analysis were used to determine the expression of LXRα in the myocardium of STZ-induced diabetic rats. The myocardial expression of LXRα target genes, long-chain acyl-CoA synthetase 3 (ACSL3), fatty acid transporter protein (FAT/CD36), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 were also detected. Bisulfite sequencing analysis was employed to examine the methylation status of the CpG island at the LXRα promoter region in the myocardium of STZ-induced diabetic rats. We found that LXRα mRNA and protein expression in the left ventricles, right ventricles, and atria of diabetic rats were gradually increased during the progression of diabetic cardiomyopathy (DCM). The mRNA expression levels of ACSL3 and FAT/CD36 and the protein expression levels of ABCA1 and ABCG1 were also markedly increased in different heart chambers of diabetic rats. Moreover, there was a significant difference in the methylation status of LXRα gene between the ventricles of control and diabetic rats (P < 0.05). Our findings suggest that elevated expression of LXRα may be involved in the progression of DCM, and demethylation of LXRα is likely to be responsible for its increased expression in myocardial tissues. Show less

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation. Show less

MicroRNAs are small non-coding RNA molecules that play essential roles in biological processes ranging from cell cycle to cell migration and invasion. Accumulating evidence suggests that miR-34a, as a key mediator of p53 tumor suppression, is aberrantly expressed in human cancers. In the present study, we aimed to explore the precise biological role of miR-34a and the global protein changes in HCC cell line HepG2 cells transiently transfected with miR-34a. Transfection of miR-34a into HepG2 cells caused suppression of cell proliferation, inhibition of cell migration and invasion. It also induced an accumulation of HepG2 cells in G1 phase. Among 116 protein spots with differential expression separated by 2-DE method, 34 proteins were successfully identified by MALDI-TOF/TOF analysis. Of these, 15 downregulated proteins may be downstream targets of miR-34a. Bioinformatics analysis produced a protein-protein interaction network, which revealed that the p53 signaling pathway and cell cycle pathway were two major hubs containing most of the proteins regulated by miR-34a. Cytoskeletal proteins such as LMNA, GFAP, MACF1, ALDH2, and LOC100129335 are potential targets of miR-34a. In conclusion, abrogation of miR-34a function could cause downstream molecules to switch on or off, leading to HCC development. Show less

Liver X receptors (LXRs) promote macrophage reverse cholesterol transport and cholesterol excretion from the body. The synthetic LXR ligands T0901317 and GW3965 were shown to significantly inhibit atherosclerosis in mice and to increase the expression of ATP-binding cassette transporter A1 (ABCA1) in the atherosclerotic lesions. However, these compounds increase plasma and hepatic triglyceride (TG) levels in mice. Methyl-3β-hydroxy-5α,6α-epoxycholanate (MHEC), synthesized from hyodeoxycholic acid, functions as an LXR agonist, but its role in atherogenesis and lipid metabolism remained to be elucidated. THP-1-derived macrophages were cultured in the medium con- taining various concentrations of MHEC or T0901317 (0-10 μmol/l) for 24 h. Reverse transcription polymerase chain reaction was used to quantify LXRα, LXRβ and ABCA1 mRNA levels in macrophages. Additionally, MHEC or T0901317 was orally administered at 10 mg/kg daily for 6 weeks in apolipoprotein E knockout (apoE⁻/⁻) mice fed a high-cholesterol diet. Plasma lipids were determined enzymatically. The area of and ABCA1 expression in the aortic atherosclerotic lesions were measured by oil red O staining and immunohistochemistry, respectively. Both MHEC and T0901317 equally stimulated LXRα and ABCA1 mRNA expression in a dose-dependent manner in THP-1-derived macrophages, but they did not induce LXRβ mRNA expression significantly. The plasma levels of total cholesterol, TG and high-density lipoprotein cholesterol were significantly higher in T0901317-treated mice than in the vehicle-treated control group. Interestingly, MHEC treatment dramatically increased plasma high-density lipoprotein cholesterol without altering plasma levels of total cholesterol and TG. Both MHEC and T0901317 equally inhibited the development of atherosclerotic lesions in apoE⁻/⁻ mice. The expression of ABCA1, a cholesterol efflux transporter, was greatly induced by the two LXR agonists in the artery wall. MHEC is a novel LXR agonist and it inhibits atherosclerosis in apoE⁻/⁻ mice without raising blood TG. Thus, MHEC relative to T0901317 may be a better therapeutic LXR agonist for the treatment of atherosclerosis. Show less

In addition to the hematopoietic effect of erythropoietin, increasing evidence suggests that erythropoietin also exerts protective effects for cardiovascular diseases. However, the role of erythropoietin and its underlying mechanism in macrophage foam cell formation are poorly understood. Compared with wild-type specimens, erythropoietin was increased in atherosclerotic aortas of apolipoprotein E-deficient (apoE(-/-)) mice, mainly in the macrophage foam cells of the lesions. Erythropoietin levels in culture medium and macrophages were significantly elevated in response to oxidized low-density lipoprotein in a dose-dependent manner. Furthermore, erythropoietin markedly attenuated lipid accumulation in oxidized low-density lipoprotein-treated macrophages, a result that was due to an increase in cholesterol efflux. Erythropoietin treatment significantly increased ATP-binding cassette transporters (ABC) A1 and ABCG1 mRNA and protein levels without affecting protein expression of scavenger receptors, including scavenger receptor-A, CD36, and scavenger receptor-BI. The upregulation of ABCA1 and ABCG1 by erythropoietin resulted from liver X receptor alpha activation, which was confirmed by its prevention on expression of ABCA1 and ABCG1 after pharmacological or small interfering RNA inhibition of liver X receptor alpha. Moreover, the erythropoietin-mediated attenuation on lipid accumulation was abolished by such inhibition. Finally, reduced lipid accumulation and marked increase in ABCA1 and ABCG1 were demonstrated in erythropoietin-overexpressed macrophages. Our data suggest that erythropoietin suppresses foam cell formation via the liver X receptor alpha-dependent upregulation of ABCA1 and ABCG1. Show less

To detect gene mutations associated with hypertrophic cardiomyopathy (HCM) in Chinese patients and possible correlations between genotype and phenotype. Twenty-one unrelated patients with hypertrophic cardiomyopathy were studied. The clinical data including symptoms, physical examination, echocardiography and electrocardiography were collected. The full ecoding exons of cardiac myosin-binding protein C gene (cMYBPC3) were amplified with PCR and the products were sequenced. Two mutations were identified in probands from two families. One mutation was frame shift mutation Pro1208fs in the exon 32 of the cMYBPC3 gene. Pro1208fs mutation was identified in a 59 years old female patient with familial hypertrophic cardiomyopathy. Symptom onset was late and a favorable clinical course was evidenced in this patient. Another mutation was missence mutation Gly507Arg in the exon 17 of the MYBPC3 gene identified in a 24 years old male patient. Diffuse thickness of left ventricular wall, impaired diastolic function and enlarged left atria were evidenced in echocardiography. No mutation was identified in the 80 control healthy individuals. cMYBPC3 might be the disease-causing genes in Chinese patients with hypertrophic cardiomyopathy. Show less

The Par3/Par6/aPKC and the CRB3/Pals1/PATJ polarity complexes are involved in regulating apical ectoplasmic specialization (ES) and blood-testis barrier (BTB) restructuring in the testis. Par6 was a component of the apical ES and the BTB. However, its level was considerably diminished at both sites at stage VIII of the cycle. Par6 also formed a stable complex with Pals1 and JAM-C (a component of the apical ES) in normal testes. When rats were treated with adjudin to induce apical ES restructuring without compromising the BTB, Par6 staining virtually disappeared at the apical ES in misaligned spermatids before their depletion. Additionally, the Par6/Pals1 complex became tightly associated with Src kinase, rendering a loss of association of the Par6/Pals1 complex with JAM-C, thereby destabilizing apical ES to facilitate spermatid loss. Primary Sertoli cell cultures with established functional BTB, but without apical ES, were next used to assess the Par6-based complex on BTB dynamics. When either Par6 or Par3 was knocked down by RNAi in Sertoli cell epithelium, a significant loss of the corresponding protein by approximately 60% in cells vs. controls was detected, alongside with a decline in aPKC after Par6, but not Par3, knockdown. This Par3 or Par6 knockdown also led to a transient loss of selected BTB proteins at the cell-cell interface, thereby compromising the BTB integrity. These findings illustrate that the Par6/Par3-based polarity complex likely coordinates the events of apical ES and BTB restructuring that take place concurrently at the opposing ends of adjacent Sertoli cells in the seminiferous epithelium during spermatogenesis. Show less

Current staging methods are inadequate for predicting the outcome of treatment of non-small-cell lung cancer (NSCLC). We developed a five-gene signature that is closely associated with survival of patients with NSCLC. We used computer-generated random numbers to assign 185 frozen specimens for microarray analysis, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, or both. We studied gene expression in frozen specimens of lung-cancer tissue from 125 randomly selected patients who had undergone surgical resection of NSCLC and evaluated the association between the level of expression and survival. We used risk scores and decision-tree analysis to develop a gene-expression model for the prediction of the outcome of treatment of NSCLC. For validation, we used randomly assigned specimens from 60 other patients. Sixteen genes that correlated with survival among patients with NSCLC were identified by analyzing microarray data and risk scores. We selected five genes (DUSP6, MMD, STAT1, ERBB3, and LCK) for RT-PCR and decision-tree analysis. The five-gene signature was an independent predictor of relapse-free and overall survival. We validated the model with data from an independent cohort of 60 patients with NSCLC and with a set of published microarray data from 86 patients with NSCLC. Our five-gene signature is closely associated with relapse-free and overall survival among patients with NSCLC. Show less

Individuals with type 1 diabetes have an increased incidence of coronary artery disease (CAD) and a higher risk of cardiovascular death compared with individuals of the same age in the general population. While chronic hyperglycaemia and insulin resistance partially explain excess CAD, little is known about the potential genetic determinants of accelerated coronary atherosclerosis in type 1 diabetes. The aim of the present study was to evaluate the association of apolipoprotein A-IV (APOA4) polymorphisms with coronary artery calcification (CAC) progression, a marker of subclinical atherosclerosis. Two previously well-studied functional APOA4 polymorphisms resulting in the substitution of the amino acid Thr for Ser at codon 347 and Gln for His at codon 360 were genotyped in 634 subjects with type 1 diabetes and 739 non-diabetic control subjects, the participants of the prospective Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. The His360 allele was associated with a significantly higher risk of CAC progression among patients with type 1 diabetes (33.7 vs 21.2%, p=0.014), but not in the control subjects (14.1 vs 11.1%, p=0.42). Logistic regression analysis confirmed that the presence of the APOA4 His360 allele predicts an increased risk of progression of coronary atherosclerosis in adults with type 1 diabetes of long duration (odds ratio = 3.3, p=0.003 after adjustment for covariates associated with CAD risk). CONCLUSIONS /INTERPRETATION: This is the first report suggesting an association between the APOA4 Gln360His polymorphism and risk of CAC progression in subjects with type 1 diabetes. Additional studies are needed to explore potential interactions between APOA4 genotypes and metabolic/oxidative stress components of the diabetic milieu leading to rapid progression of atherosclerosis. Show less

Candidate genes in inflammation, thrombosis, coagulation, and lipid metabolism pathways have been implicated in venous thromboembolism (VTE). Using DNA samples collected at baseline in the Physicians' Health Study cohort, we genotyped 92 polymorphisms from 56 candidate genes among 304 individuals who subsequently developed VTE (144 idiopathic, 156 secondary cases) and among 2070 individuals who remained free of reported vascular disease over a mean follow-up of 13.2 years to prospectively determine whether these gene polymorphisms contribute to the risk of VTE. For idiopathic VTE, in addition to the factor V (Leiden) mutation (odds ratio [OR], 5.13; 95% confidence interval [CI], 3.24 to 8.14; P<0.0001; false discovery rate [FDR], P<0.0001), an N291S lipoprotein lipase gene polymorphism (OR, 3.09; 95% CI, 1.56 to 6.09; P=0.001; FDR, P=0.036) and a Q27E beta2-adrenergic receptor gene polymorphism (OR, 1.40; 95% CI, 1.09 to 1.79; P=0.006; FDR, P=0.036) were found to be significantly associated with increased risk. For secondary VTE, a Q360H apolipoprotein A4 gene polymorphism (OR, 0.34; 95% CI, 0.18 to 0.65; P=0.001; FDR, P=0.07) and an I50V interleukin-4 receptor polymorphism (OR, 0.66; 95% CI, 0.52 to 0.84; P=0.0009; FDR, P=0.07) were moderately, but not statistically and significantly, associated with reduced risk after adjustment for multiple comparisons. These present findings are hypothesis generating and require replication and confirmation in an independent investigation. Show less

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans. Show less

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. In DN patients, triglyceride (TG) level is elevated and lipoprotein lipase (LPL) activity, which hydrolyzes TG, is decreased. The LPL S447X and apolipoprotein E (APOE) exon 4 polymorphisms affect TG levels, and the APOC3 -455T>C polymorphism affects LPL activity. Our aim was to examine the association of these polymorphisms with nephropathy in type 2 diabetes. We examined these polymorphisms in a case-control study of type 2 diabetic patients including 374 with DN and 392 without DN. LPL 447X-containing genotypes (447X+) were significantly decreased in DN patients [18.6 vs 25.6%, odds ratio (OR) = 0.66, p = 0.02], as were APOE epsilon3/epsilon3 genotypes (64.8 vs 73.1%, OR = 0.68, p = 0.01). In addition, combinations of genotypes [APOE epsilon3/epsilon3 and LPL 447X+ (OR = 0.56), APOC3 CC and LPL 447X+ (OR = 0.31), APOE epsilon3/epsilon3 and APOC3 CC (OR = 0.61] were protective for DN compared with the most common combination of the respective polymorphisms. Our findings suggest the importance of interactions among lipid genes in modulating the risk of DN. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization. Show less

Type 2 diabetes mellitus (DM) is associated with significant abnormalities of lipoprotein metabolism and coronary heart disease (CHD). The most commonly recognized lipid abnormality in type 2 DM is hypertriglyceridemia, which is known to be an independent risk factor for CHD in diabetics. The -1131T-->C polymorphism found in the newly identified apolipoprotein A5 ( APOA5 ) gene has been found to be associated with elevated plasma triglyceride (TG) concentrations in different racial groups. In this study, DNA samples from 155 control subjects, 172 type 2 diabetics and 113 type 2 DM patients with CHD were analyzed to examine the influence of APOA5 1131T-->C polymorphism on plasma lipids and the susceptibility to CHD in type 2 diabetics. The frequency of the APOA5 -1131C allele in the DM+CHD group was significantly higher than that of control subjects (37.2% vs. 27.7%, p=0.021). The distribution of the APOA5 -1131T-->C genotypes (TT, TC and CC) was 36.3%, 53.1% and 10.6% in type 2 DM patients with CHD, and 53.6%, 37.4% and 9.0% in controls, respectively (p=0.018). The frequencies of alleles and genotypes in type 2 diabetics were not significant compared to controls. In controls, plasma TG concentrations in subjects with the TT genotype were significantly lower than in those with TC/CC (0.92, 1.28 and 1.35 mmol/L for TT, TC and CC, respectively; p = 0.003 by ANOVA). These data suggest that the APOA5 -1131T-->C polymorphism might play a role in elevated plasma TG levels in type 2 diabetic patients in the Chinese population. Show less

Triglyceride-rich lipoprotein particles may promote the progression of diabetic nephropathy. Patients with diabetic nephropathy have increased plasma triglycerides and reduced activity of hepatic lipase (HL), which hydrolyzes triglycerides. We hypothesized that the HL -514C-->T polymorphism, which reduces HL expression, and its interactions with polymorphisms in apolipoprotein (apo) E and apoC3 increase the risk of diabetic nephropathy. In a case-control study involving 374 Chinese type 2 diabetic patients with and 392 without diabetic nephropathy, we genotyped the HL -514C-->T, apoE exon 4, and apoC3 -482C-->T polymorphisms. HL -514T-containing genotypes (T+) were associated with diabetic nephropathy (OR = 1.7, P = 0.0009). Adjustment by multiple logistic regression for hypertension, triglycerides, sex, non-HDL cholesterol, BMI, smoking, and alcohol intake did not diminish the association (OR = 1.8, P = 0.003). The association between HL T+ genotypes and diabetic nephropathy appeared stronger in diabetic patients with apoC3 -482 non-TT genotypes (OR = 1.9, P = 0.003) or apoE epsilon2 or epsilon4 alleles (OR = 2.2, P = 0.005). Subjects with HL TT exhibited trends toward increased triglyceride and non-HDL cholesterol levels compared with CC carriers. HL T+ genotypes might increase the risk of developing diabetic nephropathy by slowing clearance of triglyceride-rich remnant lipoproteins. In concert with other risk factors (e.g., hyperglycemia), lipid abnormalities may damage the kidneys and endothelium, where reduced binding sites for lipases may precipitate a vicious cycle of dyslipidemia, proteinuria, and nephropathy. Show less

Genetic studies in non-human primates serve as a potential strategy for identifying genomic intervals where polymorphisms impact upon human disease-related phenotypes. It remains unclear, however, whether independently arising polymorphisms in orthologous regions of non-human primates leads to similar variation in a quantitative trait found in both species. To explore this paradigm, we studied a baboon apolipoprotein gene cluster (APOA1/C3/A4/A5) for which the human gene orthologs have well-established roles in influencing plasma HDL-cholesterol and triglyceride concentrations. Our extensive polymorphism analysis of this 68 kb gene cluster in 96 pedigreed baboons identified several haplotype blocks each with limited diversity, consistent with haplotype findings in humans. To determine whether baboons, like humans, also have particular haplotypes associated with lipid phenotypes, we genotyped 634 well-characterized baboons using 16 haplotype tagging SNPs. Genetic analysis of single SNPs, as well as haplotypes, revealed an association of APOA5 and APOC3 variants with HDL-cholesterol and triglyceride concentrations, respectively. Thus, independent variation in orthologous genomic intervals does associate with similar quantitative lipid traits in both species, supporting the possibility of uncovering human quantitative trait loci genes in a highly controlled non-human primate model. Show less

To investigate the association of 21 polymorphisms within 13 genes, APOE, APOB, APOC3, CETP, LPL, PON1, MTHFR, FGB, F5, GPIIIa, SELE, ACE and AGT, with inter-individual blood pressure (BP) variation. Seven hundred and seventy-six men and 836 women, free of antihypertensive and lipid-lowering medications, were selected from the Stanislas Cohort. ANOVA on blood pressure values after adjustment for covariates [age, body mass index (BMI), contraceptive pill, tobacco and alcohol] showed that lipoprotein lipase (LPL) Ser447Ter and glycoprotein IIIA (GpIIIa) Pl polymorphisms were significantly associated with BP in women (0.01 < or = P < or = 0.05), whereas BP levels in men were significantly different according to apolipoprotein CIII (APOC3) 3206T/G and -482C/T polymorphisms (P < or = 0.05). In women, compared to the most common allele, the GpIIIa Pl allele was associated with increased mean arterial pressure (MAP) (P < 0.05) and pulse pressure (PP) (P < 0.001), and the LPL 447Ter allele was associated with decreased systolic blood pressure (SBP) and PP levels (0.001 < or = P < or = 0.05). These two polymorphisms appeared to act independently. In men, the APOC3 3206GG genotype was related to decreased diastolic blood pressure (DBP) and MAP levels (P < or = 0.01), and the APOC3 -482T allele with decreased PP levels (P < or = 0.05). The presence of both the -482C allele and the 3206GG genotype was related to decreased DBP, suggesting that specific haplotypes might be involved. The APOC3, LPL and GpIIIa genes were found to be associated with BP levels. The contributions of these genes, although modest, are consistent with the polygenic nature of BP levels. Show less

Dietary lipid acutely upregulates apolipoprotein (apo) A-IV expression by sevenfold at the pretranslational level in neonatal swine jejunum. To determine the mechanism of this regulation, two-day-old female swine received intraduodenal infusions of low- and high-triacylglycerol (TG) isocaloric diets for 24 h. Nuclear runoff assay confirmed apo A-IV gene transcriptional regulation by the high-TG diet. Footprinting analysis using the swine apo A-IV proximal promoter sequence (+14 to -246 bp) demonstrated three regions protected by the low-TG extracts. Of these three motifs, only ACCTTC showed 100% homology to the human sequence and was further studied. EMSA was performed using probes containing wild-type (WT) and mutant (M) motifs. A shift was noted with the low-TG nuclear extracts with the WT probe but not with the M probe. Excess unlabeled free WT probe competed out the shift, whereas the M probe did not. No significant shift occurred with either probe using high-TG extracts. These results suggest that a repressor protein binds to the ACCTTC motif and becomes unbound during lipid absorption, allowing transcriptional activation of the apo A-IV gene in newborn swine small intestine. Show less

Apolipoprotein A-IV (apoA-IV) has myriad functions, including roles as a post-prandial satiety factor and lipid antioxidant. ApoA-IV is expressed in mammalian small intestine and is up-regulated in response to lipid absorption. In newborn swine jejunum, a high fat diet acutely induces a 7-fold increase in apoA-IV expression. To determine whether apoA-IV plays a role in the transport of absorbed lipid, swine apoA-IV was overexpressed in a newborn swine enterocyte cell line, IPEC-1, followed by analysis of the expression of genes related to lipoprotein assembly and lipid transport, as well as quantitation of lipid synthesis and secretion. A full-length swine apoA-IV cDNA was cloned, sequenced, and inserted into a Vp and Rep gene-deficient adeno-associated viral vector, containing the cytomegalovirus immediate early promoter/enhancer and neomycin resistance gene, and was used to transfect IPEC-1 cells. Control cells were transfected with the same vector minus the apoA-IV insert. Using neomycin selection, apoA-IV-overexpressing (+AIV) and control (-AIV) clones were isolated for further study. Both undifferentiated (-D) and differentiated (+D) +AIV cells expressed 40- to 50-fold higher levels of apoA-IV mRNA and both intracellular and secreted apoA-IV protein compared with -AIV cells. Expression of other genes was not affected by apoA-IV overexpression in a manner that would contribute to enhanced lipid secretion. +D +AIV cells secreted 4.9-fold more labeled triacylglycerol (TG), 4.6-fold more labeled cholesteryl ester (CE), and 2-fold more labeled phospholipid (PL) as lipoproteins, mostly in the chylomicron/very low density lipoprotein (VLDL) density range. ApoA-IV overexpression in IPEC-1 cells enhances basolateral TG, CE, and PL secretion in chylomicron/VLDL particles. This enhancement is not associated with up-regulation of other genes involved in lipid transport. ApoA-IV may play a role in facilitating enterocyte lipid transport, particularly in the neonate receiving a diet of high fat breast milk. Show less