👤 Xiaoyin Zeng

To investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells. Human lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR. In both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells. TGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells. Show less

Ubiquitination plays important and diverse roles in modulating protein functions. As a C2-WW-HECT-type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Here, we report a novel function of Smurf1 in regulating Wnt/β-catenin signaling through targeting axin for nonproteolytic ubiquitination. Our data unambiguously demonstrate that Smurf1 ubiquitinates axin through Lys 29 (K29)-linked polyubiquitin chains. Unexpectedly, Smurf1-mediated axin ubiquitination does not lead to its degradation but instead disrupts its interaction with the Wnt coreceptors LRP5/6, which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/β-catenin signaling. The inhibitory function of Smurf1 on Wnt/β-catenin signaling is further evidenced by analysis with Smurf1 knockout murine embryonic fibroblasts. We next identified K789 and K821 in axin as the ubiquitination sites by Smurf1. Consistently, Smurf1 could neither disrupt the interaction of an axin(K789/821R) double mutant with LRP5/6 nor attenuate the phosphorylation of LRP6 in axin(K789/821R)-expressing cells. Collectively, our studies uncover Smurf1 as a new regulator for the Wnt/β-catenin signaling pathway via modulating the activity of axin. Show less

Studies on spermatogonial stem cells (SSCs) are of unusual significance because they are the unique stem cells that transmit genetic information to subsequent generations and they can acquire pluripotency to become embryonic stem-like cells that have therapeutic applications in human diseases. MicroRNAs (miRNAs) have recently emerged as critical endogenous regulators in mammalian cells. However, the function and mechanisms of individual miRNAs in regulating SSC fate remain unknown. Here, we report for the first time that miRNA-20 and miRNA-106a are preferentially expressed in mouse SSCs. Functional assays in vitro and in vivo using miRNA mimics and inhibitors reveal that miRNA-20 and miRNA-106a are essential for renewal of SSCs. We further demonstrate that these two miRNAs promote renewal at the post-transcriptional level via targeting STAT3 and Ccnd1 and that knockdown of STAT3, Fos, and Ccnd1 results in renewal of SSCs. This study thus provides novel insights into molecular mechanisms regulating renewal and differentiation of SSCs and may have important implications for regulating male reproduction. Show less

Neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) are both major serosanguinous maculopathies among the Asian elderly. They are similar in phenotype. Genetic variants in high-density lipoprotein (HDL) pathway were discovered to be associated with AMD in two genome-wide association studies. In this study with a Chinese Han cohort, we investigated the impacts of these genetic variants on nAMD and PCV separately. The missense coding variants and previously identified variants at LIPC, ABCA1, CETP, LPL and FADS1 loci were genotyped in 157 nAMD patients, 250 PCV patients and 204 controls without any macular abnormality. The known variants in CFH, ARMS2 and near HTRA1 were also genotyped. Fasting serum cholesterol levels were determined. The variants in CFH, ARMS2 and near HTRA1 were strongly associated with both PCV (P < 10(-6), 10(-7) and 10(-7) respectively) and nAMD (P < 10(-6), 10(-16) and 10(-17) respectively). None of the studied HDL-related variants were significantly associated with nAMD. A missense variant in CETP, rs5882, was significantly associated with PCV (P = 2.73 × 10(-4)). The rs5882 GG genotype had a 3.53-fold (95% CI: 1.93-6.45) increased risk for PCV, and conferred a significantly lower serum HDL-cholesterol level for PCV patients than the AA genotype (P = 0.048). These results suggest the need to separate PCV from nAMD in association studies especially with Asian cohorts, and that the HDL pathway may involve in the pathogenesis of PCV and nAMD differently. Show less

The rs17145738 single nucleotide polymorphism (SNP) near MLX interacting protein-like/transducin (beta)-like 2 (MLXIPL/TBL2) loci is associated with serum lipid levels, but the results are inconsistent in diverse ethnic/racial groups. The current study was to investigate the association of MLXIPL/TBL2 rs17145738 SNP and several environmental factors with serum lipid profiles in the Guangxi Mulao and Han populations. A total of 649 subjects of Mulao nationality and 712 participants of Han nationality aged 16-84 years were randomly selected from our previous stratified randomized samples. Genotyping was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Serum apolipoprotein (Apo) B levels were higher in Mulao than in Han (P < 0.001). There were no significant differences in the genotypic and allelic frequencies of the MLXIPL/TBL2 rs17145738 SNP between the two ethnic groups or between males and females. The T allele carriers had higher triglyceride (TG) and ApoB levels in Mulao, and higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in Han than the T allele non-carriers (P < 0.05 for all). Subgroup analyses showed that the T allele carriers had higher ApoB levels in both Mulao and Han females than the T allele non-carriers, but the T allele carriers had lower ApoB levels in Han males than the T allele non-carriers (P < 0.05, respectively). The T allele carriers in Han had higher TC, high-density lipoprotein cholesterol (HDL-C) levels and ApoA1/ApoB ratio and lower TG levels in males, and higher LDL-C levels and lower ApoA1/ApoB ratio in females than the T allele non-carriers (P < 0.05 for all). Serum TC levels in the combined population of the two ethnic groups and in Han, and HDL-C levels in Han males were correlated with genotypes (P < 0.05 for all). Serum lipid parameters were also correlated with several environmental factors (P < 0.05-0.01). The association of MLXIPL/TBL2 rs17145738 SNP and serum lipid profiles is different between the Mulao and Han populations. There is a sex-specific association in the both ethnic groups. Show less

MicroRNAs (miRNAs) emerge as new important regulators of lipid homeostasis by regulating corresponding genes. MiR-613 is a newly discovered microRNA, of which the biological function is unknown. A recent report has shown that miR-613 downregulates liver X receptor α (LXRα), a ligand-activated nuclear receptor playing an important role in the regulation of lipid metabolism. The purpose of this study is to explore the effect and the molecular basis of miR-613 on lipogenesis in HepG2 cells. HepG2 cells were transiently transfected with miR-613 mimic or control microRNA. Real time PCR, Western blot, Luciferase reporter assay and Oil Red O staining were employed to examine the expression of LXRα and its target genes involved in lipogenesis, binding site for miR-613 in 3'-untranslated region (3'-UTR) of LXRα mRNA and lipid droplet accumulation in the cells. MiR-613 dramatically suppressed the expression of LXRα and its target genes including sterol-regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), carbohydrate responsive element-binding protein (ChREBP) and acetyl-CoA carboxylase (ACC). Reporter assay showed that miR-613 directly bound to 3'-UTR of LXRα mRNA. Moreover, miR-613 significantly repressed LXRα-induced lipid droplet accumulation in HepG2 cells. Ectopic expression of LXRα without 3'-UTR markedly attenuated the miR-613-mediated downregulation of LXRα's target genes and LXRα-induced lipid droplet accumulation. MiR-613 suppresses lipogenesis by directly targeting LXRα in HepG2 cells, suggesting that miR-613 may serve as a novel target for regulating lipid homeostasis. Show less

Atherosclerosis is a chronic immunoinflammatory disease associated with blood lipid disorders. Previous studies in mice have demonstrated that liver X receptor (LXR)‑ATP‑binding cassette (ABC) A1/ABCG1/C‑C chemokine receptor type 7 (CCR7) and nuclear factor κB (NF‑κB) signaling pathways are important for atherosclerotic plaque formation. In addition, Sirtuin 1 (SIRT1) has been reported as a key regulator in the protection from risk of atherosclerosis. However, the exact mechanism by which SIRT1 prevents atherosclerosis remains largely unknown. To explore the possible mechanisms, the expression of SIRT1 and the association between SIRT1, LXR and NF‑κB in the process of foam cell formation was investigated in an in vitro human mononuclear U937 cell line. Monocyte‑derived foam cells were induced by palmitate and Ox‑LDL treatment. Oil Red O staining revealed an accumulation of a large number of lipid droplets in foam cells. Results of reverse transcription polymerase chain reaction (RT-PCR) revealed that SIRT1 expression was downregulated during foam cell formation. In addition, the expression of LXRα and its targets, ABCA1, ABCG1 and CCR7, were downregulated. However, NF‑κB and its targets, tumor necrosis factor α (TNFα) and interleukin (IL)‑1β, were upregulated in foam cells. Following activation of SIRT1 by SRT1720, the expression of LXRα and its targets increased, whereas expression of NF‑κB and its targets decreased. Furthermore, the formation of foam cells was blocked. The SIRT1 inhibitor, nicotinamide, was found to eliminate the effects of SRT1720. Results of the present study indicate that SIRT1 may prevent the formation and progression of atherosclerosis by enhancing the LXR‑ABCA1/ABCG1/CCR7 and inhibiting the NF‑κB pathways. Show less

To investigate the effects of neopterin on ABCA1 expression and cholesterol efflux in human THP-1 macrophage-derived foam cells, and to explore the role of the liver X receptor alpha (LXRα) involved. In the present study, THP-1 cells were pre-incubated with ox-LDL to become foam cells. The protein and mRNA expression were examined by Western blot assays and real-time quantitative PCR, respectively. Liquid scintillation counting and high performance liquid chromatography assays were used to test cellular cholesterol efflux and cholesterol content. Neopterin decreased ABCA1 expression and cholesterol efflux in a time- and concentration-dependent manner in THP-1 macrophage-derived foam cells, and the LXRα siRNA can reverse the inhibitory effects induced by neopterin. Neoterin has a negative regulation on ABCA1 expression via the LXRα signaling pathway, which suggests the aggravated effects of neopterin on atherosclerosis. Show less

Liver X receptor α (LXRα) plays a critical role in the transcriptional control of lipid metabolism. LXR activation induces the expression of lipogenic genes, which promote hepatic steatosis and steatohepatitis. However, the regulation of LXR is not fully understood. MicroRNAs (miRs) are regarded as important negative regulators of gene expression. In this study, we found that miR-1/miR-206 repressed LXRα-induced accumulation of lipid droplets in hepatocytes. In addition, bioinformatic analysis predicted a same putative target-site for miR-1/miR-206 located within the 3'-untranslated region (3'-UTR) of LXRα mRNA. The reporter assay revealed that miR-1/miR-206 directly targeted the 3'-UTR of LXRα mRNA. Furthermore, miR-1/miR-206 repressed LXRα expression at both mRNA and protein levels, accompanied with the inhibition of expression of LXRα target genes, such as sterol-regulatory element binding protein 1c, fatty acid synthase, carbohydrate responsive element-binding protein and acetyl-CoA carboxylase 1, which are important effectors of LXRα implicated in lipogenesis. Moreover, ectopic expression of LXRα without the 3'-UTR dramatically attenuated the miR-1/miR-206-induced decrease of lipogenic genes and lipid droplet accumulation. Taken together, we for the first time demonstrated that miR-1/miR-206 attenuated LXRα-induced lipogenesis by targeting the 3'-UTR of LXRα mRNA, suggesting that miR-1/miR-206-LXRα pathway may be a novel target for the treatment of lipogenesis-associated diseases. Show less

A dynamic balance between targeted transport and endocytosis is critical for polarized cell growth. However, how actin-mediated endocytosis is regulated in different growth modes remains unclear. Here we report differential regulation of cortical actin patch dynamics between the yeast and hyphal growth in Candida albicans. The mechanism involves phosphoregulation of the endocytic protein Sla1 by the cyclin-dependent kinase (CDK) Cdc28-Cln3 and the actin-regulating kinase Prk1. Mutational studies of the CDK phosphorylation sites of Sla1 revealed that Cdc28-Cln3 phosphorylation of Sla1 enhances its further phosphorylation by Prk1, weakening Sla1 association with Pan1, an activator of the actin-nucleating Arp2/3 complex. Sla1 is rapidly dephosphorylated upon hyphal induction and remains so throughout hyphal growth. Consistently, cells expressing a phosphomimetic version of Sla1 exhibited markedly reduced actin patch dynamics, impaired endocytosis, and defective hyphal development, whereas a nonphosphorylatable Sla1 had the opposite effect. Taken together, our findings establish a molecular link between CDK and a key component of the endocytic machinery, revealing a novel mechanism by which endocytosis contributes to cell morphogenesis. Show less

Mammalian spermatogenesis is a process whereby male germ-line stem cells (spermatogonial stem cells) divide and differentiate into sperm. Although a great deal of progress has been made in the isolation and characterization of spermatogonial stem cells (SSCs) in rodents, little is known about human SSCs. We have recently isolated human G protein-coupled receptor 125 (GPR125)-positive spermatogonia and GDNF family receptor alpha 1 (GFRA1)-positive spermatogonia using a 2-step enzymatic digestion and magnetic-activated cell sorting (MACS) from adult human testes. Cell purities of isolated human GPR125- and GFRA1-positive spermatogonia after MACS are greater than 95%, and cell viability is over 96%. The isolated GPR125- and GFRA1-positive spermatogonia coexpress GPR125, integrin, alpha 6 (ITGA6), THY1 (also known as CD90), GFRA1, and ubiquitin carboxyl-terminal esterase L1 (UCHL1), markers for rodent or pig SSCs/progenitors, suggesting that GPR125- and GFRA1-positive spermatogonia are phenotypically the SSCs in human testis. Human GPR125-positive spermatogonia can be cultured for 2 weeks with a remarkable increase in cell number. Immunocytochemistry further reveals that GPR125-positive spermatogonia can be maintained in an undifferentiated state in vitro. Collectively, the methods using enzymatic digestion and MACS can efficiently isolate and purify SSCs from adult human testis with consistent and high quality. The ability of isolating and characterizing human SSCs could provide a population of stem cells with high purity for mechanistic studies on human SSC self-renewal and differentiation as well as potential applications of human SSCs in regenerative medicine. Show less

Essential tremor (ET) is shown an autosomal dominant mode of inheritance, with no disease-causing gene has been found. Genetic variations in the leucine-rich repeat and lg domain containing nogo receptor-interacting protein genes (LINGO1 and LINGO2) were reported to be associated with an increased risk of developing ET. To explore whether the LINGO4 gene (a homologous gene of the LINGO1 and the LINGO2 genes) plays a role in ET susceptibility, we performed genetic analysis of coding region of the LINGO4 gene in 100 patients with ET from Mainland China. Two nucleotide variants had been identified: (1) T > A transition (rs61746299), predicted to lead to the amino acid change Thr444Ser, and (2) C > T transition (rs1521179), located 12 bp downstream to the end of coding region. To evaluate whether these variants are related to ET susceptibility, we investigated a total of 150 Chinese Han ET patients (77 familial ET and 73 sporadic ET) and 300 sex, age and ethnicity matched normal controls. No significant differences in genotypic and allele distributions between patients and control subjects for rs61746299 and rs1521179 (p = 0.531 and p = 0.867 for genotypic distributions; p = 1.000 and p = 0.844 for allele distributions) were observed, suggesting variants in coding region of the LINGO4 gene may play litter or no role in the risk of ET susceptibility. Show less

Endothelin-1 (ET-1), predominantly produced by vascular endothelial cells (VECs), plays an important role in the pathogenesis of inflammatory diseases. Liver X receptor (LXR), a typical nuclear receptor, is known for inhibiting expression of inflammatory molecules. However, it remains unclear whether LXR suppresses ET-1 expression. In the present study, we showed that pretreatment with GW3965, a specific ligand of LXR, significantly attenuated lipopolysaccharide (LPS)-induced ET-1 in mice plasma. The in vitro experiments showed that both LXRα and β were expressed in human VECs, and they are functional as demonstrated by induction of the target gene ABCA1 after treatment with GW3965. Moreover, activation of LXR with GW3965 in human VECs dramatically attenuated the basal and LPS-stimulated ET-1 production at both transcriptional and translational levels. Luciferase reporter assays indicated that LXR activation suppressed the transcriptional activity of the human ET-1 gene promoter, and repressed the activity of a heterologous promoter driven by the response elements of activator-1 (AP-1) or nuclear factor-κB (NF-κB). Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that activation of LXR reduced the binding of the transcriptional factors AP-1 and NF-κB to the ET-1 gene promoter region. In conclusion, activation of LXR represses ET-1 expression in vivo and in vitro, which may be involved in the negatively interfering with AP-1/NF-κB signaling. These results suggest that LXRs may serve as a novel molecular target for modulating ET-1 expression in VECs, and even for the treatment of ET-1-associated inflammatory diseases. Show less

Histone lysine acetylation and methylation have an important role during gene transcription in a chromatin context. Knowledge concerning the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains. Recently, a tandem plant homeodomain (PHD) finger (PHD1-PHD2, or PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate gene transcription during heart and muscle development, was reported to bind histones H3 and H4 in an acetylation-sensitive manner, making it the first alternative to bromodomains for acetyl-lysine binding. Here we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b highlight the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine-14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of the mouse DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription. Show less

LINGO-1 (leucine-rich repeat and Ig domain-containing, Nogo receptor-interacting protein) is an important component of the NgR receptor complex involved in RhoA activation and axon regeneration. The authors report on passive immunization with LINGO-1 polyclonal antiserum, a therapeutic approach to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). The intrathecally administered high-titer rabbit-derived antiserum can be detected around the injury site within a wide time window; it blocks LINGO-1 in vivo with high molecular specificity. In this animal model, passive immunization with LINGO-1 antiserum significantly decreased RhoA activation and increased neuronal survival. Adult rats immunized in this manner show recovery of certain hindlimb motor functions after dorsal hemisection of the spinal cord. Thus, passive immunotherapy with LINGO-1 polyclonal antiserum may represent a promising repair strategy following acute SCI. Show less

Previously, a genome-wide scan has identified a nonsynonymous single nucleotide polymorphism (rs3812316, G771C, Gln241His) in the MLXIPL gene that is associated with the level of plasma triglycerides. However, no data are available on the association of this polymorphism with coronary artery disease (CAD) in the Chinese population. The aim of this study was to evaluate the association between a gene polymorphism related to triglyceride metabolism and CAD. The genotype of the polymorphism in the MLXIPL gene was determined in 352 CAD patients and 152 CAD-free subjects. All of the participants were selected to study the MLXIPL gene rs3812316 polymorphism using the polymerase chain reaction restriction fragment length polymorphism method. In Chinese participants, we observed that there was a significant difference in genotype between the cases and controls (p = 0.002). After allowance for potential confounders, unconditional logistic analysis revealed that the SNP was significantly related to a risk in CAD patients (adjusted OR 2.96, 95% CI 1.30-5.08; p =0.004). We also found that there was a significant association between the single nucleotide polymorphism and plasma triglyceride levels (OR 1.28, 95% CI 1.061-1.542; p < 0.05). The gene sequence variation in the MLXIPL gene may serve as a novel genetic marker for the risk of significant CAD. Show less

The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible. We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant. The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression. Show less

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors. Show less

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling. Show less

Familial combined hyperlipidemia (FCH) is the most common genetic lipid disorder with an undefined genetic etiology. Apolipoprotein A5 gene (APOA5) variants were previously shown to contribute to FCH. The aim of the present study was to evaluate the association of APOA5 variants with FCH and its related phenotypes in Dutch FCH patients. Furthermore, the effects of variants in the APOA5 gene on carotid intima-media thickness (IMT) and cardiovascular disease (CVD) were examined. The study population consisted of 36 Dutch families, including 157 FCH patients. Two polymorphisms in the APOA5 gene (-1131T>C and S19W) were genotyped. Haplotype analysis of APOA5 showed an association with FCH (p=0.029), total cholesterol (p=0.031), triglycerides (p<0.001), apolipoprotein B (p=0.011), HDL-cholesterol (p=0.013), small dense LDL (p=0.010) and remnant-like particle cholesterol (p=0.001). Compared to S19 homozygotes, 19W carriers had an increased risk of FCH (OR=1.6 [1.0-2.6]; p=0.026) and a more atherogenic lipid profile, reflected by higher triglyceride (+22%) and apolipoprotein B levels (+5%), decreased HDL-cholesterol levels (-7%) and an increased prevalence of small dense LDL (16% vs. 26%). In carriers of the -1131C allele, small dense LDL was more prevalent than in -1131T homozygotes (29% vs. 16%). No association of the APOA5 gene with IMT and CVD was evident. In Dutch FCH families, variants in the APOA5 gene are associated with FCH and an atherogenic lipid profile. Show less

To better understand the pathophysiologic mechanisms underlying spinal nerve root injury induced by lumbar disk herniation (LDH), comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with LDH (the experiment group) and the otherwise healthy patients who had had implants removed from healed fractures in the lower limbs (the control group) was carried out using 2-DE followed by LC-IT-MS and database searching. Image analysis of silver-stained 2-DE gels revealed that 15 protein spots showed significant differential expression between the two groups of CSF samples (p < 0.05). After searching the database we found that in CSF of LDH patients, the expression of cystatin C, apolipoprotein A-IV, vitamin D-binding protein, neurofilament triplet L protein, IgG, tetranectin, and hemoglobin were elevated. However, ProSAAS, prostagladin D2 synthase, creatine kinase B, superoxide dismutase 1 and peroxiredoxin 2 were decreased. The subsequent ELISA measured the concentration of tetranectin, vitamin D-binding protein and cystatin C and confirmed the results of proteomic analysis. These identified proteins are involved in the pathophysiological process of spinal nerve root injury caused by herniated lumbar disk. The functional implications of the alterations in the levels of these proteins are discussed in this paper. Show less

ApoAV, a newly discovered apolipoprotein, plays a key role in human triglyceride homeostasis; however, the structure-function correlation of apoAV is not clearly understood. To explore the relationship, wild type and six deletion mutants, that is (AV (Delta(1-51)), AV (Delta(51-128)), AV (Delta(132-188)), AV (Delta(192-238)), AV (Delta(246-299)), AV (Delta(301-343))), of human apoAV expressed in Escherichia coli were studied. All the deleted regions together encompass almost the entire 343 amino acid sequence of wild type apoAV. Circular dichroism spectroscopy showed that the alpha helical content of lipid-free wild type apoAV was 46%. In comparison with wild type apoAV, AV (Delta(192-238)) and AV (Delta(301-343)) displayed significantly decreased lipid binding activities, confirming the importance of these two regions in lipid binding function of apoAV. While, the LPL activation function of apoAV remarkably impaired after deletion of residues 192-238. These findings suggested that the domain (192-238) is absolutely necessary for apoAV in lipid binding and lipoprotein lipase activation. Show less

Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease. The canonical Wnt/beta-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6), whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of beta-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/beta-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway. Show less

LDL receptor related proteins 5 and 6 (LRP5/6) and their Drosophila homolog Arrow are single-span transmembrane proteins essential for Wnt/beta-catenin signaling, likely via acting as Wnt coreceptors. How Wnt activates LRP5/6/Arrow to initiate signal transduction is not well defined. Here we show that a PPPSP motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, is necessary and sufficient to trigger Wnt/beta-catenin signaling. A single PPPSP motif, upon transfer to the LDL receptor, fully activates the Wnt pathway, inducing complete axis duplication in Xenopus and TCF/beta-catenin-responsive transcription in human cells. We further show that Wnt signal-ing stimulates, and requires, phosphorylation of the PPPSP motif, which creates an inducible docking site for Axin, a scaffolding protein controlling beta-catenin stability. Our study identifies a critical signaling module and a key phosphorylation-dependent activation step of the Wnt receptor complex and reveals a unifying logic for transmembrane signaling by Wnts, growth factors, and cytokines. Show less

To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsim) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. Exposure of tBHP 100 micromol/L to neurons for 60 min resulted in DYm loss and cytochrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis. After removal of tBHP and then further treatment with curcumin (2.5-20 micromol/L) for 18 h, curcumin abrogated Deltapsim loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family. Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly. Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. Curcumin may attenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mitochondria from oxidative damage. Show less

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3. Show less

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that the regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity. Show less

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians. Show less