👤 Houlin Wei

Genome-wide single nucleotide polymorphism (SNP) data from 936 bipolar disorder (BD) individuals and 940 psychiatrically healthy comparison individuals of North European descent were analyzed for copy number variation (CNV). Using multiple CNV calling algorithms, and validating using in vitro molecular analyses, we identified CNVs implicating several candidate genes that encode synaptic proteins, such as DLG1, DLG2, DPP6, NRXN1, NRXN2, NRXN3, SHANK2, and EPHA5, as well as the neuronal splicing regulator RBFOX1 (A2BP1), and neuronal cell adhesion molecule CHL1. We have also identified recurrent CNVs on 15q13.3 and 16p11.2-regions previously reported as risk loci for neuropsychiatric disorders. In addition, we performed CNV analysis of individuals from 215 BD trios and identified de novo CNVs involving the NRXN1 and DRD5 genes. Our study provides further evidence of the occasional involvement of genomic mutations in the etiology of BD, however, there is no evidence of an increased burden of CNVs in BD. Further, the identification of CNVs at multiple members of the neurexin gene family in BD individuals, supports the role of synaptic disruption in the etiology of BD. Show less

This study aimed to investigate the role of fatty acid synthase (FASN) in the epithelial-mesenchymal transition (EMT) of breast cancer cells. MCF-7 cells and MCF-7 cells overexpressing mitogen-activated protein kinase 5 (MCF-7-MEK5) were used in this study. MCF-7-MEK5 cells showed stable EMT characterized by increased vimentin and decreased E-cadherin expression. An In vivo animal model was established using the orthotopic injection of MCF-7 or MCF-7-MEK5 cells. Real-time quantitative PCR and western blotting were used to detect the expression levels of FASN and its downstream proteins liver fatty acid-binding protein (L-FABP) and VEGF/VEGFR-2 in both in vitro and in vivo models (nude mouse tumor tissues). In MCF-7-MEK5 cells, significantly increased expression of FASN was associated with increased levels of L-FABP and VEGF/VEGFR-2. Cerulenin inhibited MCF-7-MEK5 cell migration and EMT, and reduced FASN expression and down-stream proteins L-FABP, VEGF, and VEGFR-2. MCF-7-MEK5 cells showed higher sensitivity to Cerulenin than MCF-7 cells. Immunofluorescence revealed an increase of co-localization of FASN with VEGF on the cell membrane and with L-FABP within MCF-7-MEK5 cells. Immunohistochemistry further showed that increased percentage of FASN-positive cells in the tumor tissue was associated with increased percentages of L-FABP- and VEGF-positive cells and the Cerulenin treatment could reverse the effect. Altogether, our results suggest that FASN is essential to EMT possibly through regulating L-FABP, VEGF and VEGFR-2. This study provides a theoretical basis and potential strategy for effective suppression of malignant cells with EMT. Show less

Liver X receptor-α (LXR-α) which belongs to the nuclear receptor superfamily, is a ligand-activated transcription factor. Best known for its ability to regulate lipid metabolism and transport, LXRs have recently also been implicated in regulation of inflammatory response. The aim of this study was to investigate the preventive effects of synthetic LXR-α agonist T0901317 on LPS-induced mastitis in mice. The mouse model of mastitis was induced by injection of LPS through the duct of mammary gland. T0901317 was injected 1h before and 12h after induction of LPS intraperitoneally. The results showed that T0901317 significantly attenuated the infiltration of neutrophilic granulocytes, and the activation of myeloperoxidase (MPO); down-regulated the level of pro-inflammatory mediators including TNF-α, IL-1β, IL-6, COX-2 and PEG2; inhibited the phosphorylation of IκB-α and NF-κB p65, caused by LPS. Moreover, we report for the first time that LXR-α activation impaired LPS-induced mastitis. Taken together, these data indicated that T0901317 had protective effect on mastitis and the anti-inflammatory mechanism of T0901317 on LPS induced mastitis in mice may be due to its ability to inhibit NF-κB signaling pathway. LXR-α activation can be used as a therapeutic approach to treat mastitis. Show less

Cyanidin-3-O-β-glucoside (C3G), a typical anthocyanin pigment that exists in the human diet, has been reported to have anti-inflammatory properties. The aim of this study was to detect the effect of C3G on LPS-induced acute lung injury and to investigate the molecular mechanisms. Acute lung injury was induced by intratracheal administration of LPS in mice. Alveolar macrophages from mice were stimulated with LPS and were treated with C3G. Our results showed that C3G attenuated lung histopathologic changes, myeloperoxidase (MPO) activity, TNF-α, IL-1β and IL-6 production in LPS-induced acute lung injury model. In vitro, C3G dose-dependently inhibited TNF-α, IL-1β, IL-6, IL-10 and IFN-β production, as well as NF-κB and IRF3 activation in LPS-stimulated alveolar macrophages. Furthermore, C3G disrupted the formation of lipid rafts by depleting cholesterol and inhibited TLR4 translocation into lipid rafts. Moreover, C3G activated LXRα-ABCG1-dependent cholesterol efflux. Knockout of LXRα abrogated the anti-inflammatory effects of C3G. In conclusion, C3G has a protective effect on LPS-induced acute lung injury. The promising anti-inflammatory mechanisms of C3G is associated with up-regulation of the LXRα-ABCG1 pathway which result in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts, thereby suppressing TLR4 mediated inflammatory response. Show less

The liver X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) pathway and LXR-modulated cytokines play an important role in macrophages which mediate lipid engulfment and the inflammatory response, and participate in the process of atherosclerosis. Therefore, lipid-lowering and anti-inflammatory therapy through the activation of the LXRα/ABCA1 pathway and LXRα-modulated cytokines may prove to be one of the main treatment strategies for atherosclerosis. Alisma Decoction (AD) has long been used in China to clinically treat cardiovascular and cerebral diseases; however, the precise mechanisms involved remain to be elucidated. In the present study, we evaluated the regulation of lipids and the anti-inflammatory effects exerted by AD and investigated the underlying molecular mechanisms using oxidized low-density lipoprotein (ox-LDL)-stimulated foam cells derived from rat peritoneal macrophages. We first found that AD markedly relieved lipid deposition in foam cells as it increased LXRα and ABCA1 expression and decreased the ox-LDL-induced expression of inflammatory cytokines, such as matrix metalloproteinase-9 and interleukin-1β. Collectively, our findings suggest that blocking lipid deposition and inhibiting inflammatory response through the activation of the LXRα pathway may be one of the main mechanisms through which AD exerts its anti-atherosclerotic effects. Show less

Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. In this study, Oil-red O staining, cholesterol and triglyceride assay, Dil-oxidized LDL (oxLDL) binding assay, cholesterol efflux assay, real-time RT-PCR and Western blot analysis were used for in vitro experiments. Apolipoprotein E-deficient (apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient (apoE(-/-) iNOS(-/-) ) mice were as our in vivo models. Treatment with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, exacerbated oxLDL-induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP-binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR-A), CD36, ABCG1, or SR-B1 levels. This SNAP-mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP-downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)-dependent transcriptional regulation. Moreover, genetic deletion of iNOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1, and SR-BI in aortas and retarded atherosclerosis in apoE(-/-) mice. Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages. Show less

The Crumbs (Crb) complex, formed by Crb, PALS1, and PATJ, is evolutionarily conserved in metazoans and acts as a master cell-growth and -polarity regulator at the apical membranes in polarized epithelia. Crb intracellular functions, including its direct binding to PALS1, are mediated by Crb's highly conserved 37-residue cytoplasmic tail. However, the mechanistic basis governing the highly specific Crb-PALS1 complex formation is unclear, as reported interaction between the Crb tail (Crb-CT) and PALS1 PSD-95/DLG/ZO-1 (PDZ) domain is weak and promiscuous. Here we have discovered that the PDZ-Src homolgy 3 (SH3)-Guanylate kinase (GK) tandem of PALS1 binds to Crb-CT with a dissociation constant of 70 nM, which is ∼ 100-fold stronger than the PALS1 PDZ-Crb-CT interaction. The crystal structure of the PALS1 PDZ-SH3-GK-Crb-CT complex reveals that PDZ-SH3-GK forms a structural supramodule with all three domains contributing to the tight binding to Crb. Mutations disrupting the tertiary interactions of the PDZ-SH3-GK supramodule weaken the PALS1-Crb interaction and compromise PALS1-mediated polarity establishment in Madin-Darby canine kidney (MDCK) cysts. We further show that specific target binding of other members of membrane-associated guanylate kinases (MAGUKs) (e.g., CASK binding to neurexin) also requires the presence of their PDZ-SH3-GK tandems. Show less

Autophagy, a highly conserved process conferring cytoprotection against stress, contributes to the progression of cerebral ischemia. β-arrestins are multifunctional proteins that mediate receptor desensitization and serve as important signaling scaffolds involved in numerous physiopathological processes. Here, we show that both ARRB1 (arrestin, β 1) and ARRB2 (arrestin, β 2) were upregulated by cerebral ischemic stress. Knockout of Arrb1, but not Arrb2, aggravated the mortality, brain infarction, and neurological deficit in a mouse model of cerebral ischemia. Accordingly, Arrb1-deficient neurons exhibited enhanced cell injury upon oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Deletion of Arrb1 did not affect the cerebral ischemia-induced inflammation, oxidative stress, and nicotinamide phosphoribosyltransferase upregulation, but markedly suppressed autophagy and induced neuronal apoptosis/necrosis in vivo and in vitro. Additionally, we found that ARRB1 interacted with BECN1/Beclin 1 and PIK3C3/Vps34, 2 major components of the BECN1 autophagic core complex, under the OGD condition but not normal conditions in neurons. Finally, deletion of Arrb1 impaired the interaction between BECN1 and PIK3C3, which is a critical event for autophagosome formation upon ischemic stress, and markedly reduced the kinase activity of PIK3C3. These findings reveal a neuroprotective role for ARRB1, in the context of cerebral ischemia, centered on the regulation of BECN1-dependent autophagosome formation. Show less

PTEN is post-translationally modified by ubiquitin via association with multiple E3 ubiquitin ligases, including NEDD4-1, XIAP, and WWP2. Despite the rapid progress made in researching the impact of ubiquitination on PTEN function, our understanding remains fragmented. Building on the previously observed interaction between SIPL1 and PTEN, we report here that SIPL1 promotes PTEN polyubiquitination via lysine 48 (K48)-independent polyubiquitin chains. Substitution of the K48 residue of ubiquitin with arginine (R) enhanced SIPL1-mediated PTEN polyubiquitination. In contrast, the K63R substitution significantly reduced it. The ubiquitin-like (UBL) domain is required for SIPL1-induced PTEN polyubiquitination. This post-translational modification promoted the association of SIPL1 with PTEN. Elevated amounts of the SIPL1/PTEN complex were precipitated in 293T cells co-transfected with PTEN, SIPL1, and ubiquitin compared to cells co-transfected with SIPL1 and PTEN only. Additionally, formation of the SIPL1/PTEN complex was inhibited when either lysine-less (K0) ubiquitin or K63R ubiquitin was co-transfected together with SIPL1+PTEN. The PTEN component in the SIPL1/PTEN complex contained polyubiquitin chains. The ubiquitination reaction may play a structural role, stabilizing the SIPL1/PTEN complex, as a ubiquitin binding-defective SIPL1 mutant (TFLV) is proficient in PTEN association. Collectively, we demonstrate that SIPL1 binds PTEN and enhances PTEN polyubiquitination which in turn promotes the interaction between SIPL1 and PTEN. Show less

Sox2 is a key factor for maintaining embryonic stem cell (ESS) pluripotency, but little is known about its posttranslational regulation. Here we present evidence that the precise level of Sox2 proteins in ESCs is regulated by a balanced methylation and phosphorylation switch. Set7 monomethylates Sox2 at K119, which inhibits Sox2 transcriptional activity and induces Sox2 ubiquitination and degradation. The E3 ligase WWP2 specifically interacts with K119-methylated Sox2 through its HECT domain to promote Sox2 ubiquitination. In contrast, AKT1 phosphorylates Sox2 at T118 and stabilizes Sox2 by antagonizing K119me by Set7 and vice versa. In mouse ESCs, AKT1 activity toward Sox2 is greater than that of Set7, leading to Sox2 stabilization and ESC maintenance. In early development, increased Set7 expression correlates with Sox2 downregulation and appropriate differentiation. Our study highlights the importance of a Sox2 methylation-phosphorylation switch in determining ESC fate. Show less

Local interactions between neighbouring SNPs are hypothesized to be able to capture variants missing from genome-wide association studies (GWAS) via haplotype effects but have not been thoroughly explored. We have used a new high-throughput analysis tool to probe this underexplored area through full pair-wise genome scans and conventional GWAS in diastolic and systolic blood pressure and six metabolic traits in the Northern Finland Birth Cohort 1966 (NFBC1966) and the Atherosclerosis Risk in Communities study cohort (ARIC). Genome-wide significant interactions were detected in ARIC for systolic blood pressure between PLEKHA7 (a known GWAS locus for blood pressure) and GPR180 (which plays a role in vascular remodelling), and also for triglycerides as local interactions within the 11q23.3 region (replicated significantly in NFBC1966), which notably harbours several loci (BUD13, ZNF259 and APOA5) contributing to triglyceride levels. Tests of the local interactions within the 11q23.3 region conditional on the top GWAS signal suggested the presence of two independent functional variants, each with supportive evidence for their roles in gene regulation. Local interactions captured 9 additional GWAS loci identified in this study (3 significantly replicated) and 73 from previous GWAS (24 in the eight traits and 49 in related traits). We conclude that the detection of local interactions requires adequate SNP coverage of the genome and that such interactions are only likely to be detectable between SNPs in low linkage disequilibrium. Analysing local interactions is a potentially valuable complement to GWAS and can provide new insights into the biology underlying variation in complex traits. Show less

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1β (HP1β) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1β binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1β in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1β interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification. Show less

Plasma lipid levels are important risk factors for cardiovascular disease and are influenced by genetic and environmental factors. Recent genome wide association studies (GWAS) have identified several lipid-associated loci, but these loci have been identified primarily in European populations. In order to identify genetic markers for lipid levels in a Chinese population and analyze the heterogeneity between Europeans and Asians, especially Chinese, we performed a meta-analysis of two genome wide association studies on four common lipid traits including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) in a Han Chinese population totaling 3,451 healthy subjects. Replication was performed in an additional 8,830 subjects of Han Chinese ethnicity. We replicated eight loci associated with lipid levels previously reported in a European population. The loci genome wide significantly associated with TC were near DOCK7, HMGCR and ABO; those genome wide significantly associated with TG were near APOA1/C3/A4/A5 and LPL; those genome wide significantly associated with LDL were near HMGCR, ABO and TOMM40; and those genome wide significantly associated with HDL were near LPL, LIPC and CETP. In addition, an additive genotype score of eight SNPs representing the eight loci that were found to be associated with lipid levels was associated with higher TC, TG and LDL levels (P = 5.52 × 10(-16), 1.38 × 10(-6) and 5.59 × 10(-9), respectively). These findings suggest the cumulative effects of multiple genetic loci on plasma lipid levels. Comparisons with previous GWAS of lipids highlight heterogeneity in allele frequency and in effect size for some loci between Chinese and European populations. The results from our GWAS provided comprehensive and convincing evidence of the genetic determinants of plasma lipid levels in a Chinese population. Show less

Previous biological studies showed evidence of a genetic link between obesity and pigmentation in both animal models and humans. Our study investigated the individual and joint associations between obesity-related single nucleotide polymorphisms (SNPs) and both human pigmentation and risk of melanoma. Eight obesity-related SNPs in the FTO, MAP2K5, NEGR1, FLJ35779, ETV5, CADM2, and NUDT3 genes were nominally significantly associated with hair color among 5,876 individuals of European ancestry. The genetic score combining 35 independent obesity-risk loci was significantly associated with darker hair color (beta-coefficient per ten alleles = 0.12, P value = 4 × 10(-5)). However, single SNPs or genetic scores showed non-significant association with tanning ability. We further examined the SNPs at the FTO locus for their associations with pigmentation and risk of melanoma. Among the 783 SNPs in the FTO gene with imputation R (2) quality metric >0.8 using the 1,000 genome data set, ten and three independent SNPs were significantly associated with hair color and tanning ability respectively. Moreover, five independent FTO SNPs showed nominally significant association with risk of melanoma in 1,804 cases and 1,026 controls. But none of them was associated with obesity or in linkage disequilibrium with obesity-related variants. FTO locus may confer variation in human pigmentation and risk of melanoma, which may be independent of its effect on obesity. Show less

Paeonol, a phenolic component purified from Paeonia suffruticosa (Cortex Moutan), is used in traditional Chinese medicine to treat inflammatory diseases. However, little is known about the effect of paeonol on cholesterol metabolism. We investigated the efficacy of paeonol on cholesterol metabolism and the underlying mechanism in macrophages and apolipoprotein E deficient (apoE(-/-)) mice. Treatment with paeonol markedly attenuated cholesterol accumulation induced by oxidized LDL in macrophages, which was due to increased cholesterol efflux. Additionally, paeonol enhanced the mRNA and protein expression of ATP-binding membrane cassette transport protein A1 (ABCA1) but did not alter the protein level of ABCG1 or other scavenger receptors. Inhibition of ABCA1 activity with a pharmacological inhibitor, neutralizing antibody or small interfering RNA (siRNA), negated the effects of paeonol on cholesterol efflux and cholesterol accumulation. Furthermore, paeonol induced the nuclear translocation of liver X receptor α (LXRα) by increasing its activity. siRNA knockdown of LXRα abolished the paeonol-induced upregulation of ABCA1, promotion of cholesterol efflux and suppression of cholesterol accumulation. Moreover, atherosclerotic lesions, hyperlipidemia and systemic inflammation were reduced and the protein expression of ABCA1 was increased in aortas of paeonol-treated apoE(-/-) mice. Paeonol may alleviate the formation of foam cells by enhancing LXRα-ABCA1-dependent cholesterol efflux. Show less

The transient receptor potential vanilloid type 1 (TRPV1) is crucial in the pathogenesis of atherosclerosis; yet its role and underlying mechanism in the formation of macrophage foam cells remain unclear. Here, we show increased TRPV1 expression in the area of foamy macrophages in atherosclerotic aortas of apolipoprotein E-deficient mice. Exposure of mouse bone-marrow-derived macrophages to oxidized low-density lipoprotein (oxLDL) upregulated the expression of TRPV1. In addition, oxLDL activated TRPV1 and elicited calcium (Ca(2+)) influx, which were abrogated by the pharmacological TRPV1 antagonist capsazepine. Furthermore, oxLDL-induced lipid accumulation in macrophages was ameliorated by TRPV1 agonists but exacerbated by TRPV1 antagonist. Treatment with TRPV1 agonists did not affect the internalization of oxLDL but promoted cholesterol efflux by upregulating the efflux ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Moreover, the upregulation of ABC transporters was mainly through liver X receptor α-(LXRα-) dependent regulation of transcription. Moreover, the TNF-α-induced inflammatory response was alleviated by TRPV1 agonists but aggravated by the TRPV1 antagonist and LXR α siRNA in macrophages. Our data suggest that LXR α plays a pivotal role in TRPV1-activation-conferred protection against oxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages. Show less

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis. Show less

Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC. Show less

HEY bHLH transcription factors have been shown to regulate multiple key steps in cardiovascular development. They can be induced by activated NOTCH receptors, but other upstream stimuli mediated by TGFß and BMP receptors may elicit a similar response. While the basic and helix-loop-helix domains exhibit strong similarity, large parts of the proteins are still unique and may serve divergent functions. The striking overlap of cardiac defects in HEY2 and combined HEY1/HEYL knockout mice suggested that all three HEY genes fulfill overlapping function in target cells. We therefore sought to identify target genes for HEY proteins by microarray expression and ChIPseq analyses in HEK293 cells, cardiomyocytes, and murine hearts. HEY proteins were found to modulate expression of their target gene to a rather limited extent, but with striking functional interchangeability between HEY factors. Chromatin immunoprecipitation revealed a much greater number of potential binding sites that again largely overlap between HEY factors. Binding sites are clustered in the proximal promoter region especially of transcriptional regulators or developmental control genes. Multiple lines of evidence suggest that HEY proteins primarily act as direct transcriptional repressors, while gene activation seems to be due to secondary or indirect effects. Mutagenesis of putative DNA binding residues supports the notion of direct DNA binding. While class B E-box sequences (CACGYG) clearly represent preferred target sequences, there must be additional and more loosely defined modes of DNA binding since many of the target promoters that are efficiently bound by HEY proteins do not contain an E-box motif. These data clearly establish the three HEY bHLH factors as highly redundant transcriptional repressors in vitro and in vivo, which explains the combinatorial action observed in different tissues with overlapping expression. Show less

Molecular and cellular signaling pathways are involved in the process of neural differentiation from human embryonic stem cells (hESC) to terminally differentiated neurons. The Sonic hedgehog (SHH) morphogen is required to direct the differentiation of hESC to several neural subtypes, for example, dopaminergic (DA) or motor neurons. However, the roles of SHH signaling and the pathway target genes that regulate the diversity of cellular responses arising from SHH activation during neurogenesis of hESC have yet to be elucidated. In this study, we report that overexpression of SHH in hESC promotes the derivation of neuroprogenitors (NP), increases proliferation of NP, and subsequently increases the yield of DA neurons. Next, gene expression changes resulting from the overexpression of SHH in hESC-derived NP were examined by genome-wide transcriptional profiling. Categorizing the differentially expressed genes according to the Gene Ontology biological processes showed that they are involved in numerous cellular processes, including neural development, NP proliferation, and neural specification. In silico GLI-binding sites analysis of the differentially expressed genes also identified a set of putative novel direct target genes of SHH in hESC-derived NP, which are involved in nervous system development. Electrophoretic mobility shift assays and promoter-luciferase assays confirmed that GLI1 binds to the promoter region and activates transcription of HEY2, a NOTCH signaling target gene. Taken together, our data provide evidence for the first time that there is cross-talk between the NOTCH and SHH signaling pathways in hESC-derived NP and also provide significant new insights into transcriptional targets in SHH-mediated neural differentiation of hESC. Show less

Mitogen/extracellular signal-regulated kinase kinase-5 (MEK5), which belongs to a network of mitogen-activated protein kinase pathways, play a pivotal role in carcinogenesis. The purpose of this study was to investigate whether variants in the MEK5 gene promoter were involved in susceptivity of individuals to sporadic colorectal cancer (CRC). In the present hospital-based case-control study of 737 patients with sporadic CRC and 703 healthy control subjects in a southern Chinese population, the two polymorphisms of MEK5 promoter (i.e., rs7172582C>T and rs3743354T>C) were genotyped by TaqMan assay. There were significant differences between cases and controls in the genotype and allele distribution of the MEK5 gene rs3743354T>C polymorphism. The rs3743354 CC genotype was associated with a significantly decreased risk of CRC when compared with the TT genotype (adjusted odds ratios [ORs]=0.43; 95% confidence interval [CI], 0.24-0.77). Compared to the T allele, a significant correlation was detected between the presence of the C allele and decreased risk of CRC (adjusted OR=0.79; 95% CI, 0.61-0.94). The decreased risk of CRC associated with rs3743354 variant genotypes (i.e., CT+CC) was found in the smoker subgroup (adjusted OR=0.63; 95% CI=0.45-0.88). Further, environmental factors, including smoking and drinking, interacted with rs3743354C variant genotypes to reduce CRC risk. Western blot analysis showed that the levels of MEK5 protein in sporadic CRC neoplastic tissues and adjacent normal colorectal epithelium tissues were lower in the carriers of rs3743354 CC genotypes than that in those with rs3743354 TT genotypes or those with rs3743354 TC genotypes. However, no significant association was found between the rs7172582C>T polymorphism and risk of CRC. These data indicate that the rs3743354 polymorphism in the MEK5 promoter may affect the risk of developing CRC. Show less

A link between fructose drinking and nonalcoholic fatty liver disease (NAFLD) has been demonstrated in human and rodent animals. The aim of the present study was to investigate whether endoplasmic reticulum (ER) stress is mediated in the development of fructose-induced NAFLD. Female CD-1 mice were fed with 30% fructose solution for eight weeks. Hepatic lipid accumulation was assessed. Hepatic nuclear sterol regulatory element-binding protein (SREBP)-1c was measured. Results showed that hepatic SREBP-1c was activated in mice fed with fructose solution. Fatty acid synthase (fas) and acetyl-CoA carboxylase (acc), two target genes of SREBP-1c, were up-regulated. Fructose-evoked hepatic SREBP-1c activation seemed to be associated with insulin-induced gene (Insig)-1 depletion. An ER stress and unfolded protein response (UPR), as determined by an increased glucose-regulated protein (GRP78) expression and an increased eIF2α and PERK phosphorylation, were observed in liver of mice fed with fructose solution. Phenylbutyric acid (PBA), an ER chemical chaperone, not only significantly attenuated ER stress, but also alleviated fructose-induced hepatic Insig-1 depletion. PBA inhibited fructose-evoked hepatic SREBP-1c activation and the expression of SREBP-1c target genes, and protected against hepatic lipid accumulation. In conclusion, ER stress contributes, at least in part, to hepatic SREBP-1c activation and lipid accumulation in fructose-evoked NAFLD. Show less

To study the essence of chronic viral hepatitis B (CHB) of damp-heat retention in the middle-jiao syndrome (DRMS) from plasma proteomic angle. Plasma proteomic analyses of plasma whole protein of patients in the group with CHB of DRMS (20 cases) and subjects in the health control group (5 cases) were compared using two-dimensional gel electrophoresis (2-DE), mass spectrography, and other bioinformatics analyses methods. Eight protein dots with obvious regularity changes of differential expression were obtained by 2-DE. Seven protein dots were obtained by mass spectrography (One protein dot with undetected results): apolipoprotein C2 (APO-C2), vitronectin (VN), haptoglobin (HPT), transthyretin (TTHY), APO-A1, serum amyloid P-component (SAMP), and APO-A4. Compared with the health control group, the expressions of APO-A1 and APO-A4 were somewhat higher and the expressions of the expressions of the rest five protein dots were obviously down-regulated. APO-Al and APO-A4 were of potential significance in the diagnosis of CHB patients of DRMS, prognostic markers, or treatment targets. Show less

Leber congenital amaurosis (LCA) is one of the most severe eye dystrophies characterized by severe vision loss at an early stage and accounts for approximately 5% of all retinal dystrophies. The purpose of this study was to identify a novel LCA disease allele or gene and to develop an approach combining genetic mapping with whole exome sequencing. Three patients from King Khaled Eye Specialist Hospital (KKESH205) underwent whole genome single nucleotide polymorphism genotyping, and a single candidate region was identified. Taking advantage of next-generation high-throughput DNA sequencing technologies, whole exome capture sequencing was performed on patient KKESH205#7. Sanger direct sequencing was used during the validation step. The zebrafish model was used to examine the function of the mutant allele. A novel missense mutation in Bardet-Biedl syndrome 4 protein (BBS4) was identified in a consanguineous family from Saudi Arabia. This missense mutation in the fifth exon (c.253G>C;p.E85Q) of BBS4 is likely a disease-causing mutation as it segregates with the disease. The mutation is not found in the single nucleotide polymorphism (SNP) database, the 1000 Genomes Project, or matching normal controls. Functional analysis of this mutation in zebrafish indicates that the G253C allele is pathogenic. Coinjection of the G253C allele cannot rescue the mislocalization of rhodopsin in the retina when BBS4 is knocked down by morpholino injection. Immunofluorescence analysis in cell culture shows that this missense mutation in BBS4 does not cause obvious defects in protein expression or pericentriolar localization. This mutation likely mainly reduces or abolishes BBS4 function in the retina. Further studies of this allele will provide important insights concerning the pleiotropic nature of BBS4 function. Show less

To investigate the relationship of NOR-1 with the inhibition of inflammatory reaction in mice Kupffer cells (KCs) induced by lipopolysaccharide (LPS) via liver X receptor alpha (LXR alpha). KCs from male KM mice were isolated by density gradient centrifugation, incubated and then randomly assigned to three groups: control group, LPS treated group and LPS+T0901317 treated group. The mRNA and protein expressions of LXR alpha and NOR-1 in each group were determined by RT-PCR, immunofluorescent assay and western blot, respectively. The densities of TNF alpha and IL-10 in supernatants were evaluated by enzyme linked immunosorbent assay (ELISA). The mRNA and protein expression levels of LXR alpha in LPS + T0901317 group were the highest as compared to the other two groups (0.748+/-0.072 and 1.217+/-0.133 respectively), The mRNA and protein expression levels of NOR-1 in LPS+ T0901317 group were the highest as compared to the other two groups (2.726+/-0.065 and 0.842+/-0.058 respectively). The densities of supernatant TNF alpha in LPS group and IL-10 in LPS+T0901317 group were the highest [(450.89+/-78.52) ng/L and (537.41+/-36.41) ng/L respectively]. Promoting the expression of LXR alpha in KCs can elevate the NOR-1 expression and then inhibit inflammatory reaction. Show less

ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50=0.09 μM). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPARγ; EC50=5.2-9.3 μM). Liver X receptor α (LXRα) is a target gene of PPARγ and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXRα protein expression in HepG2 cells. Addition of PPARγ antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXRα protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPARγ. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPARγ-LXRα-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism. 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

Fatty acid metabolism is perturbed in atherosclerotic lesions, but whether it affects lesion formation is unknown. To determine whether fatty acid synthesis affects atherosclerosis, we inactivated fatty-acid synthase (FAS) in macrophages of apoE-deficient mice. Serum lipids, body weight, and glucose metabolism were the same in FAS knock-out in macrophages (FASKOM) and control mice, but blood pressure was lower in FASKOM animals. Atherosclerotic extent was decreased 20-40% in different aortic regions of FASKOM as compared with control mice on Western diets. Foam cell formation was diminished in FASKOM as compared with wild type macrophages due to increased apoAI-specific cholesterol efflux and decreased uptake of oxidized low density lipoprotein. Expression of the anti-atherogenic nuclear receptor liver X receptor alpha (LXRalpha; Nr1h3) and its downstream targets, including Abca1, were increased in FASKOM macrophages, whereas expression of the potentially pro-atherogenic type B scavenger receptor CD36 was decreased. Peroxisome proliferator-activated receptor alpha (PPARalpha) target gene expression was decreased in FASKOM macrophages. PPARalpha agonist treatment of FASKOM and wild type macrophages normalized PPARalpha target gene expression as well as Nr1h3 (LXRalpha). Atherosclerotic lesions were more extensive when apoE null mice were transplanted with LXRalpha-deficient/FAS-deficient bone marrow as compared with LXRalpha-replete/FAS-deficient marrow, consistent with anti-atherogenic effects of LXRalpha in the context of FAS deficiency. These results show that macrophage FAS deficiency decreases atherosclerosis through induction of LXRalpha and suggest that FAS, which is induced by LXRalpha, may generate regulatory lipids that cause feedback inhibition of LXRalpha in macrophages. Show less

Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by formation of benign cartilage-capped tumors (exostoses), typically located at the juxtaepiphyseal regions of long bones. It is genetically heterogeneous with at least three chromosomal loci: EXT1 on 8q24.1, EXT2 on 11p11, and EXT3 on 19p. EXT1 and EXT2 have been cloned and are responsible for over 80% of cases. A Chinese family with HME has been analyzed in the present study. Linkage analysis was firstly performed to determine which of the three EXT genes could be the candidate gene, then mutation screening by PCR and direct sequencing was carried out. A novel nonsense mutation (c.1006C>T) in exon 6 of EXT2, which converts the codon CAA (Gln) to the stop codon (TAA) (Gln336X), was identified. Next, prenatal diagnosis was performed and the pregnancy was determined to be normal. A new EXT2 nonsense mutation was found in a Chinese family with hereditary multipe exostoses. The information was used for a case of prenatal diagnosis. Show less