👤 Yu Huang

Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117-137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex. Show less

Myosin binding protein C (MYBPC3) plays a role in ventricular relaxation. The aim of the study was to investigate the association between cardiac myosin binding protein C (MYBPC3) gene polymorphisms and diastolic heart failure (DHF) in a human case-control study. A total of 352 participants of 1752 consecutive patients from the National Taiwan University Hospital and its affiliated hospital were enrolled. 176 patients diagnosed with DHF confirmed by echocardiography were recruited. Controls were matched 1-to-1 by age, sex, hypertension, diabetes, renal function and medication use. We genotyped 12 single nucleotide polymorphisms (SNPs) according to HapMap Han Chinese Beijing databank across a 40 kb genetic region containing the MYBPC3 gene and the neighboring DNA sequences to capture 100% of haplotype variance in all SNPs with minor allele frequencies ≥ 5%. We also analyzed associations of these tagging SNPs and haplotypes with DHF and linkage disequilibrium (LD) structure of the MYBPC3 gene. In a single locus analysis, SNP rs2290149 was associated with DHF (allele-specific p = 0.004; permuted p = 0.031). The SNP with a minor allele frequency of 9.4%, had an odds ratio 2.14 (95% CI 1.25-3.66; p = 0.004) for the additive model and 2.06 for the autosomal dominant model (GG+GA : AA, 95% CI 1.17-3.63; p = 0.013), corresponding to a population attributable risk fraction of 12.02%. The haplotypes in a LD block of rs2290149 (C-C-G-C) was also significantly associated with DHF (odds ratio 2.10 (1.53-2.89); permuted p = 0.029). We identified a SNP (rs2290149) among the tagging SNP set that was significantly associated with early DHF in a Chinese population. Show less

Panax notoginseng (Burk.) F.H. Chen has been used as a health product and natural remedy in traditional medicine for cardiovascular diseases for more than 1000 years in Asia, including China, Japan, and Korea. Panax notoginseng saponins (PNS) are the major effective ingredients extracted from Panax notoginseng. The purpose of this study was to investigate whether Panax notoginseng saponins (PNS) attenuated atherosclerosis by inducing liver X receptor alpha (LXRα) expression and to elucidate the mechanisms responsible for the effects. The AS rats were treated once daily with PNS (100 mg/kg, i.p.), and pathological changes in the aorta were observed using Sudan IV staining. The expression of LXRα in the aortic wall was measured by Western blot analysis. THP-1 macrophages were cultured with PNS in the presence or absence of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRα antagonist. The expression of LXRα and its target genes ATP-binding cassette A1 and G1 (ABCA1, ABCG1) were determined by qRT-PCR. The transcriptional activation of the LXRα gene promoter was analyzed by a reporter assay. The NF-κB DNA binding activity and the expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1) was evaluated respectively by Trans-AM NF-κB ELISA and ELISA in THP-1 macrophages that were stimulated with LPS after treatment with PNS and GGPP. PNS treatment alleviated the typical pathological changes associated with atherosclerosis in rats. The expression of LXRα was increased in rat aortas after treatment with PNS. In vitro, PNS increased LXRα mRNA levels in THP-1 macrophages. The reporter assays showed that PNS enhanced transcriptional activation of the LXRα gene promoter and led to the upregulation of ABCA1 and ABCG1 expression. This upregulation could be reversed by treatment with GGPP. Additionally, PNS inhibited NF-κB DNA binding activity and reduced secretion of IL-6 and MCP-1 in LPS-stimulated THP-1 macrophages. These effects could be reversed by GGPP. The results indicated that the PNS-mediated attenuation of AS may, at least partly, due to LXRα uprergulation. The mechanisms of action included enhancement transcriptional activation of the LXRα gene promoter by PNS and subsequent upregulation of ABCA1 and ABCG1 and inhibition of NF-κB DNA binding activity. Show less

Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice. Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice. Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells. Show less

Both inflammation and cholesterol accumulation play important roles in the development of non-alcoholic fatty liver disease. This study was undertaken to investigate whether inflammation aggravated cholesterol accumulation via disrupting hepatic cholesterol export and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, and tumor necrosis factor alpha (TNF-α) stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammation. Intracellular cholesterol level was examined by Oil Red O staining and quantitative analysis. Bile acid level was quantified by colorimetric analysis. (3)[H] cholesterol assay by scintillation counting was performed to evaluate the cholesterol efflux. The mRNA and protein expression was examined by real-time polymerase chain reaction and Western blot. Inflammation increased cholesterol accumulation in livers of C57BL/6J mice and in HepG2 cells. High-fat diet in mice and low-density lipoprotein (LDL) loading in HepG2 cells increased bile acid synthesis and cholesterol efflux, enhanced the mRNA and protein expression of liver X receptor α (LXRα), peroxisome proliferator-activated receptors (PPARα, γ), cholesterol 7α-hydroxylase (CYP7A1) and ATP-binding cassette transporter A1 (ABCA1). However, inflammation reduced bile acid synthesis and cholesterol efflux even in high-fat-diet-fed mice and HepG2 cells in the presence of LDL loading. The enhanced effects of these genes and proteins expression due to high-fat diet and LDL loading were inhibited by inflammation both in vivo and in vitro. Inflammation disrupted PPAR-LXR-CYP7A1/ABCA1-mediated bile acid synthesis and cholesterol efflux resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells. Show less

Studies were done on analysis of biological processes in the same high expression (fold change ≥2) activated PTHLH feedback-mediated cell adhesion gene ontology (GO) network of human hepatocellular carcinoma (HCC) compared with the corresponding low expression activated GO network of no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection). Activated PTHLH feedback-mediated cell adhesion network consisted of anaphase-promoting complex-dependent proteasomal ubiquitin-dependent protein catabolism, cell adhesion, cell differentiation, cell-cell signaling, G-protein-coupled receptor protein signaling pathway, intracellular transport, metabolism, phosphoinositide-mediated signaling, positive regulation of transcription, regulation of cyclin-dependent protein kinase activity, regulation of transcription, signal transduction, transcription, and transport in HCC. We proposed activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network. Our hypothesis was verified by the different activated PTHLH feedback-mediated cell adhesion GO network of HCC compared with the corresponding inhibited GO network of no-tumor hepatitis/cirrhotic tissues, or the same compared with the corresponding inhibited GO network of HCC. Activated PTHLH coupling feedback phosphoinositide to G-protein receptor signal-induced cell adhesion network included BUB1B, GNG10, PTHR2, GNAZ, RFC4, UBE2C, NRXN3, BAP1, PVRL2, TROAP, and VCAN in HCC from GEO dataset using gene regulatory network inference method and our programming. Show less

Inflammation plays important roles in the development of diabetic retinopathy (DR). How Müller cells contribute to DR-related inflammation remains unclear. We hypothesized that under diabetic conditions, elevated histone acetylations in Müller cells contribute to the inflammatory response. In this study, significantly increased histone acetylations, elevated histone acetyltranferases levels, and decreased histone deacetylases levels were found in the retinas of diabetic rats. Elevated AcH3K9 and AcH3K18 were partially co-stained with Müller cells on retinal sections by immunofluorescence staining. Consistently, high-glucose (HG) treated rMC-1 cells, a Müller cell line, also showed upregulation of acetylated histones, accompanied with the overexpression of GFAP, p-STAT3, and NFκB-p65, and two inflammatory genes, TNFα and MCP-1. Meanwhile, sodium butyrate (NaB)-induced upregulation of acetylated histones is also accompanied with transcription of inflammatory genes. Minocycline, a drug with beneficial effects on DR, was found to downregulate HG-induced Müller cell activation, inflammation, and acetylated H3K18 bound to the promoters of GFAP and inflammatory genes by chromatin immunoprecipitation assay. Furthermore, the effects of minocycline on HG-induced elevation in histone acetylations were also demonstrated in isolated primary rat Müller cells. These findings suggest the elevation of histone acetylations in Müller cells plays important regulating roles in the inflammatory response during diabetic conditions. Inhibition of histone acetylation by minocycline is a novel function that may contribute to its beneficial effects on DR. Show less

Endometrial carcinoma is one of the most common gynecological malignancies in women. The diagnosis of the disease at early or premalignant stages is crucial for the patient's prognosis. To date, diagnosis and follow-up of endometrial carcinoma and hyperplasia require invasive procedures. Therefore, there is considerable demand for the identification of biomarkers to allow non-invasive detection of these conditions. In this study, we performed a quantitative proteomics analysis on serum samples from simple endometrial hyperplasia, complex endometrial hyperplasia, atypical endometrial hyperplasia, and endometrial carcinoma patients, as well as healthy women. Serum samples were first depleted of high-abundance proteins, labeled with isobaric tags (iTRAQ), and then analyzed via two-dimensional liquid chromatography and tandem mass spectrometry. Protein identification and quantitation information were acquired by comparing the mass spectrometry data against the International Protein Index Database using ProteinPilot software. Bioinformatics annotation of identified proteins was performed by searching against the PANTHER database. In total, 74 proteins were identified and quantified in serum samples from endometrial lesion patients and healthy women. Using a 1.6-fold change as the benchmark, 12 proteins showed significantly altered expression levels in at least one disease group compared with healthy women. Among them, 7 proteins were found, for the first time, to be differentially expressed in atypical endometrial hyperplasia. These proteins are orosomucoid 1, haptoglobin, SERPINC 1, alpha-1-antichymotrypsin, apolipoprotein A-IV, inter-alpha-trypsin inhibitor heavy chain H4, and histidine-rich glycoprotein. The differentially expressed proteins we discovered in this study may serve as biomarkers in the diagnosis and follow-up of endometrial hyperplasia and endometrial carcinoma. Show less

The link between lipoprotein metabolism and Alzheimer's disease (AD) has been established. Apolipoprotein A-IV (apoA-IV), a component of lipoprotein particles similar to apolipoprotein E, has been suggested to play an important role in brain metabolism. Although there are clinical debates on the function of its polymorphism in AD, the pathologic role of apoA-IV in AD is still unknown. Here, we report that genetic ablation of apoA-IV is able to accelerate AD pathogenesis in mice. In a mouse model that overexpresses human amyloid precursor protein (APP) and presenilin 1, genetic reduction of apoA-IV augments extracellular amyloid-β peptide (Aβ) burden and aggravates neuron loss in the brain. In addition, genetic ablation of apoA-IV also accelerates spatial learning deficits and increases the mortality of mice. We have found that apoA-IV colocalizes within Aβ plaques in APP/presenilin 1 transgenic mice and binds to Aβ in vitro. Subsequent studies show that apoA-IV in this model facilitates Aβ uptake in the Aβ clearance pathway mediated by astrocytes rather than the amyloidogenic pathway of APP processing. Taken together, we conclude that apoA-IV deficiency increases Aβ deposition and results in cognitive damage in the mouse model. Enhancing levels of apoA-IV may have therapeutic potential in AD treatment. Show less

Huntington's disease (HD) is a progressive neurodegenerative disease caused by an unstable CAG trinucleotide repeat expansion. The need for biomarkers of onset and progression in HD is imperative, since currently reliable outcome measures are lacking. We used two-dimensional electrophoresis and mass spectrometry to analyze the proteome profiles in cerebrospinal fluid (CSF) of 6 pairs of HD patients and controls. Prothrombin, apolipoprotein A-IV (Apo A-IV) and haptoglobin were elevated in CSF of the HD patients in comparison with the controls. We used western blot as a semi-quantified measurement for prothrombin and Apo A-IV, as well as enzyme linked immunosorbent assay (ELISA) for measurement of haptoglobin, in 9 HD patients and 9 controls. The albumin quotient (Qalb), a marker of blood-brain barrier (BBB) function, was not different between the HD patients and the controls. The ratios of CSF prothrombin/albumin (prothrombin/Alb) and Apo A-IV/albumin (Apo A-IV/Alb), and haptoglobin level were significantly elevated in HD. The ratio of CSF prothrombin/Alb significantly correlated with the disease severity assessed by Unified Huntington's Disease Rating Scale (UHDRS). The results implicate that increased CSF prothrombin, Apo A-IV, and haptoglobin may be involved in pathogenesis of HD and may serve as potential biomarkers for HD. Show less

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption. Show less

Several polymorphisms in the apolipoprotein C3 (APOC3) gene have been found association with hypertriglyceridemia(HTG), but the link with coronary heart disease(CHD) risk between ethnicities was still controversial. Among them, reseachers paid more attentions to the promoter polymorphisms T-455C and C-482T because both of them located in insulin-responsive element (IRE) and insulin was thought to exert its action by down-regulating APOC3 gene expression. The aim of this study was to investigate the association of the two polymorphisms of APOC3 with CHD in a Han population in East China. TaqMan SNP Genotyping Assays were carried out to detect the genotypes of APOC3 gene, including the T-455C and C-482T, in 286 subjects with CHD and 325 controls without CHD. The levels of serum lipid profiles were also detected by biochemical methods. There was no difference of genotype frequencies and allele frequencies between the CHD population and the controls(P > 0.05). Compared with the most common genotype -455TT or -482CC, the variants had neither significantly increased CHD risk, nor the lipid variables showed any statistically relevant differences in the research population. The adjusted OR of CHD were 5.67 [0.27-18.74] and 0.75 [0.20-2.73] in carriers of the APOC3 -455C and -482T variants, respectively(P > 0.05). There was also no significant difference in APOC3 haplotype distribution in CHD and controls, but there was a strong linkage disequilibrium between T-455C and C-482T with D' = 0.9293, 0.8881, respectively(P < 0.0001). Our data did not support a relationship between the two polymorphisms of APOC3 gene and risk of CHD in the Han population in East China. Show less

Mutations in the CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early onset neurodegenerative disorder. JNCL is the most common of the NCLs, a group of disorders with infant or childhood onset that are caused by single gene mutations. The NCLs, although relatively rare, share many pathological and clinical similarities with the more common late-onset neurodegenerative disorders, while their simple genetic basis makes them an excellent paradigm. The early onset and rapid disease progression in the NCLs suggests that one or more key cellular processes are severely compromised. To identify the functional pathways compromised in JNCL, we have performed a gain-of-function modifier screen in Drosophila. We find that CLN3 interacts genetically with the core stress signalling pathways and components of stress granules, suggesting a function in stress responses. In support of this, we find that Drosophila lacking CLN3 function are hypersensitive to oxidative stress yet they respond normally to other physiological stresses. Overexpression of CLN3 is sufficient to confer increased resistance to oxidative stress. We find that CLN3 mutant flies perceive conditions of increased oxidative stress correctly but are unable to detoxify reactive oxygen species, suggesting that their ability to respond is compromised. Together, our data suggest that the lack of CLN3 function leads to a failure to manage the response to oxidative stress and this may be the key deficit in JNCL that leads to neuronal degeneration. Show less

Dual specificity phosphatase 6 (DUSP6) is a member of the MAP kinase phophatase family. DUSP6 inactivates extracellular signal-regulated kinase (ERK), belonging to the MAP kinase family, and can act in tumor suppressive pathways. The aim of this study was to investigate associations of DUSP6 expression with expression of ERK and Ki-67 and with clinicopathological parameters in lung adenocarcinoma and squamous cell carcinoma. A total of 102 squamous cell carcinomas and 66 adenocarcinomas were studied using immunohistochemistry for DUSP6, ERK1/2, and Ki-67. In 66 adenocarcinomas, high DUSP6 expression was positively correlated with ERK1/2 expression. High DUSP6 expression was correlated with lower histological grade and lower Ki-67 index in the adenocarcinomas. In 102 squamous cell carcinomas, high DUSP6 expression was correlated with lower ERK expression, with greater smoking pack-years, but not with the Ki-67 index. These results indicate that DUSP6 acts as a negative feedback regulator of ERK in adenocarcinoma progression, but that DUSP6 does not play a role in the downregulation of ERK in squamous cell carcinoma. The differential expression of DUSP6 correlated with Ki-67 index, suggesting that DUSP6 plays an important role in cancer resistance in different subtypes of non-small cell lung carcinoma. Show less

Congenital heart disease (CHD) is the malformation of the heart during embryonic development, contributing to the inadequate function of the heart. A recently suggested gene hairy-related transcription factor 2 (HEY2), is an important determinant of mammalian heart development and functions thereby. We had preformed a direct sequencing within 768 Chinese CHD patients in the HEY2 gene. However, we did not reveal any diagnostic alterations in the coding regions by direct sequencing in HEY2, nevertheless this work expands our knowledge of the causes of CHD in the other way. Show less

Progressive axonal loss from chronic demyelination in multiple sclerosis (MS) is the key contributor to clinical decline. Failure to regenerate myelin by adult oligodendrocyte precursor cells (OPCs), a widely distributed neural stem cell population in the adult brain, is one of the major causes of axonal degeneration. In order to develop successful therapies to protect the integrity of axons in MS, it is important to identify and understand the key molecular pathways involved in myelin regeneration (remyelination) by adult OPCs. This review highlights recent findings on the critical signaling pathways associated with OPC differentiation following CNS demyelination. We discuss the role of LINGO-1, Notch, Wnt, and retinoid X receptor (RXR) signaling, and how they might be useful pharmacological targets to overcoming remyelination failure in MS. Show less

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G(i) and G(q) α-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma β-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCR-stimulated fatty acid oxidation in hepatocytes. Show less

The nuclear receptor liver X receptor (LXR) is a ligand-dependent transcription factor that plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. MicroRNAs (miRNAs) are recently recognized important negative regulators of gene expression. In this report, we showed that miRNA hsa-miR-613 played an important role in the autoregulation of the human LXRα gene. hsa-miR-613 targeted the endogenous LXRα through its specific miRNA response element (613MRE) within the LXRα 3'-untranslated region. Interestingly and paradoxically, the expression of hsa-miR-613 itself was induced upon the activation of LXR. However, hsa-miR-613 did not appear to be a direct LXR target gene. Instead, the positive regulation of hsa-miR-613 by LXR was mediated by the sterol regulatory element binding protein (SREBP)-1c, a known LXR target gene. Promoter analysis revealed an SREBP response element in the hsa-miR-613 gene promoter. Treatment with insulin also induced the expression of hsa-miR-613 in an SREBP-1c-dependent manner, further supporting the role of SREBP-1c in the positive regulation of this miRNA species. Finally, the autoinduction of LXRα by a LXR agonist was enhanced when hsa-miR-613 was inhibited or SREBP-1c was down-regulated. hsa-miR-613 appeared to specifically target the human LXRα. We propose that the negative regulation mediated by hsa-miR-613 and SREBP-1c and the previously reported positive regulation mediated by an LXR response element in the LXRα gene promoter constitute a ying-yang mechanism to ensure a tight regulation of this nuclear receptor of many metabolic functions. Show less

Craniofacial anomalies (CFAs) are the most frequently occurring human congenital disease, and a major cause of infant mortality and childhood morbidity. Although CFAs seems to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathophysiological mechanisms for most CFAs are currently unknown. Here we reveal a role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient in Wwp2 develop malformations of the craniofacial region. Wwp2 is present in cartilage where its expression is controlled by Sox9. Our studies demonstrate that Wwp2 influences craniofacial patterning through its interactions with Goosecoid (Gsc), a paired-like homeobox transcription factor that has an important role in craniofacial development. We show that Wwp2-associated Gsc is a transcriptional activator of the key cartilage regulatory protein Sox6. Wwp2 interacts with Gsc to facilitate its mono-ubiquitylation, a post-translational modification required for optimal transcriptional activation of Gsc. Our results identify for the first time a physiological pathway regulated by Wwp2 in vivo, and also a unique non-proteolytic mechanism through which Wwp2 controls craniofacial development. Show less

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation at the synovial membrane. Although great progress has been made recently in exploring the etiology and pathogenesis of RA, its molecular pathological mechanism remains to be further defined and it is still a great challenge in determining the diagnosis and in choosing the appropriate therapy in early patients. This study was performed to screen candidate RA-associated serum proteins by comparative proteomics to provide research clues to early diagnosis and treatment of RA. Sera isolated from 6 RA patients and 6 healthy volunteers were pooled respectively and high-abundance proteins were depleted by Plasma 7 Multiple Affinity Removal System. The protein expression profiles between the two groups were then compared by two-dimensional gel electrophoresis (2-DE) and the proteins over/under-expressed by more than 3-fold were identified by mass spectrometry analysis. To validate the differential expression levels of the identified proteins between the two groups, ELISA was performed in two of the identified proteins in individual sera from 32 RA patients and 32 volunteers. Eight proteins which over/under-expressed in sera of RA patients were identified. Among them, chain A of transthyretin (TTR) was under-expressed, while serum amyloid A protein, apolipoprotein A (ApoA)-IV, ApoA-IV precursor, haptoglobin 2, ceruloplasmin (Cp), immunoglobulin superfamily 22 and HT016 were over-expressed. ELISA test confirmed that Cp expressed remarkably higher while TTR obviously lower in RA group compared with volunteer group. There were 8 identified proteins differentially expressed between RA group and volunteer group, which might be candidate RA-associated proteins and might be promising diagnostic indicators or therapeutic targets for RA. Show less

to explore the potential role of apolipoprotein A5 (apoA5) on the hypertriglyceridemia (HTG)-lowering effects of statin. twenty-four Sprague-Dawley rats were randomized into 3 groups: (1) control group (n = 8), with no special treatment; (2) HTG group (n = 8), treated with 10% fructose water for 6 weeks; (3) statin group (n = 8), treated with 10% fructose water for 2 weeks and cotreated with atorvastatin 10 mg×kg(-1)×d(-1) for another 4 weeks. Body weight, fasting plasma lipids and the hepatic expressions of apoA5 and peroxisome proliferator activated receptor (PPAR)α were determined. In separate in vitro experiments, we tested the effects of atorvastatin on TG and the expressions of apoA5 and PPARα in HepG2 cells. (1) at 6 weeks, plasma TG was higher in rats in HTG group than in controls, which was significantly reduced in statin group (both P < 0.05). (2) Rat hepatic apoA5 expression in HTG group was significantly lower than in control group and was significantly higher in statin group than in HTG group (both P < 0.05). (3) Similarly, rat PPARα mRNA expression in HTG group was lower than in control group and was higher in statin group than in HTG group (both P < 0.05). (4) Statin significantly upregulated the expressions of apoA5 and PPARα and decreased TG in HepG2 cells. The above effects induced by statin was blocked in the presence of PPARα inhibitor. upregulation of apoA5 expression contributes to TG lowering effect of statin via PPARα signaling pathway. Show less

Hypertriglyceridemia is associated insulin resistance in obese people. Recently identified apolipoprotein A5 (apoA5) is involved in triglyceride (TG) metabolism. This study was to investigate the role of apoA5 in insulin resistance-related hypertriglyceridemia in obesity. 682 participants including 340 non-obese individuals and 342 obese individuals were recruited in this study. Plasma apoA5 levels were measured. The insulin resistance in participants was assessed by homeostasis model assessment of insulin resistance (HOMA-IR). An insulin resistant and hypertriglyceridemic rat model was established by high-fructose diet with obese Zucker rats as positive controls. Besides, two insulin resistant models in vitro were induced by insulin and tumor necrosis factor-alpha (TNFalpha) in HepG2 cells. Obese participants had lower plasma apoA5 levels. Plasma apoA5 levels were inversely correlated with TG, body mass index and HOMA-IR in humans. Furthermore, hepatic and plasma apoA5 reduced in fructose-fed rats whereas no significant changes of apoA5 were observed in obese Zucker rats. In addition, treatment of HepG2 cells with insulin and TNFalpha decreased apoA5 expression and increased TG content. Thus, decreased apolipoprotein A5 is implicated in insulin resistance-related hypertriglyceridemia in obesity. Show less

Effective gene therapy requires regulated gene expression and vector safety. We developed a strategy to exponentially increase native promoter activity while retaining inherent regulation by inserting multi-copy response elements (REs) into non-adjacent locations. For the hepatocyte nuclear factor (HNF) 4α-dependent Hnf1a (MODY3) gene, HNF4α stimulation increased from 5- to 90-fold by inserting 3 additional HNF4α REs (H4REs). Constructing a promoter with two 4xH4REs 0.25kb apart by duplicating the 4xH4RE fragment increased stimulation to >1000-fold. HNF4α-induced protein expression by the duplicate 4xH4RE Hnf1a promoter was comparable to a viral promoter. Converting the two Apolipoprotein C3 (ApoC3) H4REs spaced 0.61kb apart to 4xH4REs achieved a similar result. Increasing spacing to 2.1kb with non-promoter DNA abolished the augmentation. Finally, converting the HNF1α RE of the HNF4A (MODY1) P2 promoter to 4xH1RE and adding a second 4xH1RE 0.84kb upstream increased HNF1α stimulation from 26- to >200-fold. Deleting intervening DNA to produce 0.23-kb spacing increased stimulation to >500-fold. Spaced multi-copy RE motifs is a novel strategy for engineering promoters that boosts activity far beyond other techniques. Augmentation of three promoters suggests that this approach is potentially applicable to other promoters for gene therapy and might obviate the need for viral promoters. Show less

An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies. Show less

Insulin resistance results in dysregulated hepatic gluconeogenesis that contributes to obesity-related hyperglycemia and progression of type 2 diabetes mellitus (T2DM). Recent studies show that MAPK phosphatase-3 (MKP-3) promotes gluconeogenic gene transcription in hepatoma cells, but little is known about the physiological role of MKP-3 in vivo. Here, we have shown that expression of MKP-3 is markedly increased in the liver of diet-induced obese mice. Consistent with this, adenovirus-mediated MKP-3 overexpression in lean mice promoted gluconeogenesis and increased fasting blood glucose levels. Conversely, shRNA knockdown of MKP-3 in both lean and obese mice resulted in decreased fasting blood glucose levels. In vitro experiments identified forkhead box O1 (FOXO1) as a substrate for MKP-3. MKP-3-mediated dephosphorylation of FOXO1 at Ser256 promoted its nuclear translocation and subsequent recruitment to the promoters of key gluconeogenic genes. In addition, we showed that PPARγ coactivator-1α (PGC-1α) acted downstream of FOXO1 to mediate MKP-3-induced gluconeogenesis. These data indicate that MKP-3 is an important regulator of hepatic gluconeogenesis in vivo and suggest that inhibition of MKP-3 activity may provide new therapies for T2DM. Show less

Exposure to particulate matter (PM) is associated with systemic health effects, but the cellular and molecular mechanisms are unclear. We hypothesized that, if circulating mononuclear cells play an important role in mediating systemic effects of PM, they would show gene expression changes following exposure. Peripheral blood samples were collected before (0 h) and at 24 h from healthy subjects exposed to filtered air (FA) and ultrafine carbon particles (UFPs, 50 microg/m(3)) for 2 h in a previous study (n = 3 each). RNA from mononuclear cell fraction (> 85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. Selected genes were confirmed in five additional subjects from the same study. We identified 1713 genes (UFP 24 h vs. FA 0 and 24 h, P < 0.05, false discovery rate of 0.01). The top 10 upregulated genes (fold) were CDKN1C (1.86), ZNF12 (1.83), SRGAP2 (1.82), FYB (1.79), LSM14B (1.79), CD93 (1.76), NCSTN (1.70), DUSP6 (1.69), TACC1 (1.68), and H2AFY (1.68). Upregulation of CDKN1C and SRGAP2 was confirmed by real-time-PCR. We entered 1020 genes with a ratio >1.1 or <-1.1 into the Ingenuity Pathway Analysis and identified pathways related to inflammation, tissue growth and host defense against environmental insults, such as, insulin growth factor 1 signaling, insulin receptor signaling and NF-E2-related factor-2-mediated oxidative stress response pathway. Two-hour exposures to UFP produced gene expression changes in circulating mononuclear cells. These gene changes provide biologically plausible links to PM-induced systemic health effects, especially those in the cardiovascular system and glucose metabolism. Show less

Hereditary multiple exostoses (HME) is an autosomal dominantly inherited disorder characterized by multiple benign cartilage-capped exostoses. Clinical manifestation of the disease is heterogenous. Overriding toes, scoliosis, spinal cord compression, and brachydactyly caused by shortening of metatarsals are rare findings. EXT1 and EXT2 are the genes responsible in most HME patients. We have characterized 11 HME families and 6 sporadic cases involving a total of 37 patients and performed mutational analysis of EXT1 and EXT2. Structural modeling of the wild and mutant proteins was also performed. Thirteen mutations were identified, including 8 that are novel. Among the novel mutations in EXT1, c.1004T>G-associated HME exhibited overriding toes and scoliosis, c.1883+2T>A-associated HME exhibited brachydactyly, and c.459₄₆₀delCT-associated exostosis arising from vertebra T4 caused spinal cord compression. Our structural predictions revealed four domains in the proteins encoded by EXT1 and EXT2: signalP, transmembrane regions, exostosin, and glyco_transf-64. The mutations truncated either part or whole of the exostosin domain and/or the C terminus of the glyco_transf-64 domain, or occurred within one of the domains. Our results provide new data for genetic diagnosis, identification of presymptomatic carriers, phenotype-genotype correlation, and understanding of the mechanisms of disease. Show less

Multiple osteochondromas (MO) is an autosomal-dominant disorder and mutations in EXT1 and EXT2 account up to 78% of the cases studied, including missense, nonsense, frameshift, and splice-site mutations. EXT1 and EXT2 encode glycosyltransferases required for the synthesis of heparan sulfate (HS) chains. The molecular pathogenesis underlying these mutations is still largely unknown. A heterozygous c.1173 + 1G > T (EXT2) mutation was identified in a three-generation 34-member MO family and is present in all 19 affected members. The consequence of this mutation is exon 7 being spliced out, and the result is a shift in the codon-reading frame from position 360 (R360) of the amino acid sequence leading to a premature termination codon, and the mutant mRNA is degraded to an undetectable level. Interestingly, HS glycosaminoglycans were also undetectable in the cartilage cap of the tumors by immunostaining. Full penetrance of this mutation in all affected members ranging from 5 to 70 years of age suggests this primary defect in EXT2 mRNA level, in conjunction with other cellular changes such as enhanced heparanase expression, can produce profound effect on the synthesis of HS chains in cartilage, the consequence of which impacts on the regulation of chondrocyte proliferation and differentiation. Show less

Non-steroidal compounds that inhibit 17beta-hydroxysteroid dehydrogenase isoform 3 (17beta-HSD3), an enzyme catalyzing the final step in testosterone biosynthesis in Leydig cells, are under development for male contraceptive or treatment of androgen dependent diseases including prostate cancer. A series of curcumin analogues with more stable chemical structures were compared to curcumin as inhibitors of 17beta-HSD3 in rat intact Leydig cells as well as rat and human testis microsomes. Show less