👤 Siwei Mao

Ubiquitin-specific protease 22 (USP22) is a member of the "death-from-cancer" signature, which plays a key role in cancer progression. Previous evidence has shown that USP22 is overexpressed and correlates with poor prognosis in glioma. The effect and mechanism of USP22 in glioma malignancy, especially cancer stemness, remain elusive. Herein, we find USP22 is more enriched in stem-like tumorspheres than differentiated glioma cells. USP22 knockdown inhibits cancer stemness in glioma cell lines. With a cell-penetrating TAT-tag protein, B cell-specific Moloney murine leukemia virus integration site 1 (BMI1), a robust glioma stem-cell marker, is found to mediate the effect of USP22 on glioma stemness. By immunofluorescence, USP22 and BMI1 are found to share similar intranuclear expression in glioma cells. By analysis with immunohistochemistry and bioinformatics, USP22 is found to positively correlate with BMI1 at the post-translational level only rather than at the transcriptional level. By immunoprecipitation and in vivo deubiquitination assay, USP22 is found to interact with and deubiquitinate BMI1 for protein stabilization. Microarray analysis shows that USP22 and BMI1 mutually regulate a series of genes involved in glioma stemness such as POSTN, HEY2, PDGFRA and ATF3. In vivo study with nude mice confirms the role of USP22 in promoting glioma tumorigenesis by regulating BMI1. All these findings indicate USP22 as a novel deubiquitinase of BMI1 in glioma. We propose a working model of the USP22-BMI1 axis, which promotes glioma stemness and tumorigenesis through oncogenic activation. Thus, targeting USP22 might be an effective strategy to treat glioma especially in those with elevated BMI1 expression. Show less

Previous studies have revealed that women with gestational diabetes mellitus (GDM) have an increased risk of developing preeclampsia (PE). The possible reason is the abnormal lipid metabolism caused by GDM that leads to dysfunction of vascular endothelial cells and atherosclerosis, resulting in the onset of PE. However, studies focusing on the pathogenesis of PE in syncytiotrophoblast of GDM patients are lacking. This study aimed to compare differentially expressed proteins from syncytiotrophoblast between women with GDM and women with GDM with subsequently developed PE. Syncytiotrophoblast samples were obtained from pregnant women immediately after delivery. To explore the protein expression changes of syncytiotrophoblast that might explain the pathogenesis of PE in women with GDM, quantitative proteomics was performed using tandem mass tag (TMT) isobaric tags and liquid chromatography-tandem mass spectrometry. Bioinformatics analysis was performed to enrich the biological processes that these differentially expressed proteins were involved in. A total of 28,234 unique peptides and 4140 proteins were identified in all samples. Among them, 23 differentially expressed proteins were identified between patients with GDM and patients with GDM with subsequently developed PE. Therein, 11 proteins were upregulated and 12 proteins were downregulated. Two relative proteins (FLT1 and PABPC4) were independently verified using immunoblotting analysis. Bioinformatic results indicated that the onset of PE in patients with GDM is a multifactorial disorder, involving factors such as apoptosis, transcriptional misregulation, oxidative stress, lipid metabolism, cell infiltration and migration, and angiogenesis. These results indicated that the inadequacy of endometrium infiltration, angiogenic disorder, and oxidative stress in syncytiotrophoblast are more likely to occur in patients with GDM and may be the potential mechanisms leading to such patients secondarily developing severe early-onset PE. Show less

Factors that contribute to the onset of atherosclerosis may be elucidated by bioinformatic techniques applied to multiple sources of genomic and proteomic data. The results of genome wide association studies, such as the CardioGramPlusC4D study, expression data, such as that available from expression quantitative trait loci (eQTL) databases, along with protein interaction and pathway data available in Ingenuity Pathway Analysis (IPA), constitute a substantial set of data amenable to bioinformatics analysis. This study used bioinformatic analyses of recent genome wide association data to identify a seed set of genes likely associated with atherosclerosis. The set was expanded to include protein interaction candidates to create a network of proteins possibly influencing the onset and progression of atherosclerosis. Local average connectivity (LAC), eigenvector centrality, and betweenness metrics were calculated for the interaction network to identify top gene and protein candidates for a better understanding of the atherosclerotic disease process. The top ranking genes included some known to be involved with cardiovascular disease ( Show less

The present study is aimed to evaluate difference of lipid metabolism related gene single nucleotide polymorphisms (SNPs) with ischemic stroke (IS) in Han and Uighur population of Xinjiang, China. Four hundred eight patients with ischemic stroke and 347 unrelated healthy individuals of age and sex matched were genotyped for Apolipoprotein A5 (ApoA5), lipoprotein lipase (LPL), Cholesteryl ester transfer protein (CETP) and low-density lipoprotein receptor (LDL-R) genes. Their mutation difference was analyzed by SNaP shot techniques. GeneMapper4.1 SPSS20.0 software was used for data management and analysis. Using a single locus analysis, the distribution difference of genotype loci in ischemic stroke cases and controls were detected to assess the genetic risk factors of ischemic stroke. Significance differences of genotype distribution in ischemic stroke cases and controls were observed in LDLR rs688 in Han and Uighur population in recessive model from analysis of single gene locus. It also was found that dramatic difference of triglyceride (TG) of LPL rs328 and systolic blood pressure in CETP rs708277 of total population. In binary logistic regression analysis of total studied population, ischemic stroke was observed significantly associated with LDLR rs688 both addictive model (TT/CC, adjusted OR = 1.47, 95% CI = 1.04-2.07) and recessive model (TT/CT + CC, adjusted Odds ratio (OR) = 2.66, 95% Confidence Interval (CI) = 1.37-5.14). In Han population, ischemic stroke was observed significantly associated with rs688 both in addictive model (TT/CC, adjusted OR = 3.27, 95% CI = 1.06-10.05). In Uighur population, no significant association was found between gene polymorphisms and the risk of ischemic stroke. Combined analysis of multiple gene and loci, interaction effects of LDLR rs688 C/T, ApoA5 rs662799 A/G and CETP rs708272 C/T denoted a significant influence on IS susceptibility. Single nucleotide polymorphisms of lipid metabolism relative gene were significantly associated with the morbidity of ischemic stroke in Han population. The interaction effects of rs688 C/T with ApoA5 rs662799 A/G and CETP rs708272 C/T promoted the occurrence of IS. Show less

To explore the association of silent information regulator 1( SIRT1) rs12778366 and apolipoprotein C3( APOC3) rs2854116 gene polymorphisms with the susceptibility of nonalcoholic fatty liver disease( NAFLD). One hundred and thirty two NAFLD patients and 252 healthy controls were enrolled in our present study according to the ultrasound diagnosis and physical examination. Venousblood samples were obtained in the morning after an overnight fast, and the samples were used to analyze the biochemical index, relating to hepatic enzymes, blood lipid and blood glucose metabolism. DNA was extracted from whole blood, and polymerase chain reaction( PCR) and mass ARRAY were used to determine the genotypes of target genes. Compared with TT genotype carriers, the SIRT1 genotype rs12778366 increased the risk of NAFLD, with OR = 1. 126( 95% CI 0. 673-1. 886)( P > 0. 05). The APOC3rs2854116 TC + TT genotype increased the risk of NAFLD compared with CC genotype( OR = 1. 044, 95% CI 0. 601-1. 814, P > 0. 05). The adjusted odds ratios had no significant changes after adjusting for gender, age and BMI. Logistic regression showed that TG, body mass index( BMI), FPG, WC and UA were the independent risk factors for NAFLD( P < 0. 05), but the polymorphisms of SIRT1 rs12778366 and APOC3 rs2854116 had no significant relationship with the risk of NAFLD. SIRT1 rs12778366 and APOC3 rs2854116 polymorphisms were not associated with NAFLD susceptibility. Show less

To investigate whether HDL-C level in pregnant Chinese Han women of late second trimester correlated with loci in high-density lipoprotein-cholesterol (HDL-C)-related genes found in genome-wide association studies (GWAS). Seven single-nucleotide polymorphisms (rs3764261 in The following polymorphisms were statistically associated with HDL-C level after adjusting for age, gestational week, pre-pregnancy BMI and state of GDM or HOMAIR: (i) rs3764261 (b = -0.055 mmol/L, 95% CI -0.101 to -0.008, Several risk alleles found to be related to HDL-C in GWAS are also associated with HDL-C levels in pregnant Chinese Han women and these risk loci contribute additively to low HDL-C levels. Show less

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced β-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate β-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development. Show less

Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of β-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase RNF146 through an NF-κB-related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of β-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases. Show less

Hyperlipidemia has unique adverse effects on pregnant women and their offspring. The underlying genetic factors related to lipid levels in pregnant populations need more studies. This study aimed to investigate the relationship between 6 single-nucleotide polymorphisms (SNPs) and plasma lipid levels during pregnancy. A total of 2060 pregnant women were recruited. Fasting plasma lipids were measured in the third trimester of pregnancy. Six SNPs (rs1260326 in GCKR, rs1800775 in CETP, rs515135 in APOB, rs1800588 in LIPC, rs964184 in ZPR1, and and rs4420638 in APOC1 of each participant were genotyped using TaqMan allelic discrimination assays. The relationships between the 6 loci and plasma lipids were analyzed using a multiple linear regression method. Two variants, rs1260326 (β = 0.220, 95% confidence interval [CI] = 0.148-0.291; P = 2.048 × 10 Loci found by genome-wide association studies to be associated with plasma lipid levels in the general population were also related to lipid levels in the third trimester of pregnancy. This finding implies that changes to lipid profiles during gestation may be associated with SNPs of lipid-related genes. Variants of lipid-related genes are important predictors for estimating lipid concentrations during the pregnancy. Show less

The small GTPase Rab26 is involved in multiple processes, such as vesicle-mediated secretion and autophagy. However, the mechanisms and functions of Rab26 in the human pulmonary microvascular endothelial cells (HPMVECs) are not clear. In this study, we thoroughly investigated the role and novel mechanism of Rab26 in permeability and apoptosis of HPMVECs using a self-assembled Rab26 siRNA loaded DNA Y-motif nanoparticle (siRab26-DYM) and Rab26 adenovirus. We found that siRab26-DYM could be efficiently transfected into HPMVECs in a time- and dose-dependent manner. Importantly, the siRab26-DYM nanovector markedly aggravated the LPS-induced apoptosis and hyper-permeability of HPMVECs by promoting the nuclear translocation of Foxo1, and subsequent activation of Toll-like receptor 4 (TLR4) signal pathway. Overexpression of Rab26 by Rab26 adenoviruses partially inactivated LPS-induced TLR4 signaling pathway, suppressed the cell apoptosis and attenuated the hyperpermeability of HPMVECs. These results suggest that the permeability and apoptosis of HPMVECs can be modulated by manipulating Rab26 derived TLR4 signaling pathway, and that Rab26 can be potential therapeutic target for the treatment of vascular diseases related to endothelial barrier functions. Show less

17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) converts the inactive Δ4-androstenedione (A) to testosterone (T). Its deficiency is the most common testosterone biosynthesis defect that results in 46,XY Disorders Of Sex Development (DSD). However, the disease is difficult to distinguish from other 46,XY DSD for similar clinical phenotypes. Therefore, genetic testing provides good criteria for the diagnosis of the disease. In this study, HSD17B3 gene was examined in 3 unrelated Chinese patients with 46,XY DSD. Direct sequencing and quantitative PCR of HSD17B3 gene revealed the presence of a compound heterozygous mutation (p.I60T/exon1 deletion) in Patient 1, a homozygous (p.I60T) mutation in Patient 2 and a frameshift mutation (p.V25Efs∗54) and an exon1 deletion in Patient 3. All of the mutations have not been reported previously. These novel mutations may expand the mutation database of HSD17B3 gene and provide us new insights into the molecular mechanism of 17β-HSD3 deficiency. It is noteworthy that when direct sequence analysis showed a rare homozygous mutation in patients with non-consanguineous parents, "apparent homozygosity" should be taken into an account and the intragenic deletion should be screened. In addition, when single mutation was found in patients with disease in recessive heredity mode, the intragenic deletion should also be screened. Show less

The aim of the present study is to investigate whether the single nucleotide polymorphism (SNP) in lipid metabolism related genes would affect the effectiveness of atorvastatin in both Han and Uighur populations. 200 ischemic stroke patients were treated with atorvastatin. The differences of blood lipid level and their ratios were measured. Six lipid related genes, HMGCR, APOA5, LPL, CETP, LDLR and PCSK9 were selected as candidate genes. And nine SNP loci in these six genes were genotyped by SNaPshot technique. In all patients treated with atorvastatin, the SNP rs662799 significantly affected the ratio of x0394;LDL and x0394;LDL/LDL (p < 0.05); the SNP rs320 significantly affected the ratio of x0394;LDL/LDL and x0394;(LDL/HDL)/(LDL/HDL) (p < 0.01) and the SNP rs708272 significantly affected the ratio of x0394;LDL (p < 0.05). In Han population treated with atorvastatin, the SNP rs662799 significantly affected the ratio of x0394;TG (p < 0.05); the SNP rs320 significantly affected the ratio of x0394;LDL/LDL and x0394;(LDL/HDL)/(LDL/HDL) (p < 0.01). In Uighur population treated with atorvastatin, the SNP rs2266788 significantly affected the ratio of x0394;HDL (p < 0.05); the SNP rs662799 significantly affected the ratio of x0394;LDL/LDL (p < 0.05) and the SNP rs708272 significantly affected the ratio of x0394;LDL (p < 0.05). Polymorphisms of rs662799 and rs2266788 in APOA5 gene, rs320 in LPL gene and rs708272 in CETP gene had significant association with the effect of the lipid-lowering therapy via atorvastatin calcium on ischemic stroke patients. Show less

Yin-deficiency-heat (YDH) syndrome is a concept in Traditional Chinese Medicine (TCM) for describing subhealth status. However, there are few efficient diagnostic methods available for confirming YDH syndrome. To explore the novel method for diagnosing YDH syndrome, we applied iTRAQ to observe the serum protein profiles in YDH syndrome rats and confirmed protein levels by ELISA. A total of 92 differentially expressed proteins (63 upregulated proteins and 29 downregulated proteins), which were mainly involved in complement and coagulation cascades and glucose metabolism pathway, were identified by the proteomic experiments. Kininogen 1 (KNG1) was significantly increased ( Show less

CLN3 is a recently identified anti-apoptotic gene, which has been demonstrated to be highly expressed in a diverse range of cancer cell lines, including ovarian cancer. In the present study, RNA interference, mediated by a lentivirus expressing CLN3 short hairpin RNA (shRNA) was utilized to knockdown the expression of CLN3 in the A2780 human ovarian cancer cell line, and its cisplatin‑resistant and carboplatin‑resistant sublines, A2780/DDP and A2780/CBP cells. It was revealed that the mRNA and protein expression levels of CLN3 were significantly reduced in the CLN3‑specific shRNA‑transduced cells, compared with the untransduced and control shRNA‑transduced cells. In addition, specific knockdown of CLN3 in these cells inhibited cell proliferation and led to cell cycle arrest at the G0/G1 phase, with eventual apoptosis. CLN3 knockdown caused increases in the levels of Bax, FAX, cleaved‑caspase 3, cleaved‑caspase 8 and cleaved‑RARP, but decreased the level of Bcl‑2. Finally, it was observed that CLN3 depletion markedly reduced the half maximum inhibitory concentration in the A2780/DDP and A2780/CBP cells. Taken together, these data suggested that CLN3 is involved in tumorigenesis and drug resistance in ovarian cancer, and may serve as a promising therapeutic target for its treatment. Show less

The excessive proliferation of vascular smooth muscle cells was key factor in the restenosis of vein graft. And the Notch signaling was demonstrated to regulate vSMC proliferation and differentiation. Soluble Jagged-1 (sJag1) can inhibit Notch signaling in vitro and in vivo; however, its capacity to suppress restenosis of vein graft remains unknown. Under the microscope, the left jugular vein of these rats was interposed into the left common carotid artery, followed without any treatment (control), or with Ad-Jag1 (treatment) or placebo (DMSO) post operation. We showed that Ad-Jag1 can attenuate restenosis of vein graft by inducing decreased proliferation and increased apoptosis in vivo. Notch1-Hey2 signaling is critical for the development of intima thickening by controlling vSMC-fate determination. By blocking Notch signaling, Ad-Jag1 can significantly inhibit intima thickening. These studies identify that Ad-Jag1 can restore the vSMC phenotype and inhibit the vSMC proliferation by suppression of Notch1 signaling, and thus open a new avenue for the treatment of restenosis in vein graft. Show less

Hepatic resection is the most curative treatment option for early-stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early-stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high-copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer-specific and recurrence-free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti-FGF19 treatment in these patients. Show less

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling. Show less

Pediatric osteosarcoma is characterized by multiple somatic chromosomal lesions, including structural variations (SVs) and copy number alterations (CNAs). To define the landscape of somatic mutations in pediatric osteosarcoma, we performed whole-genome sequencing of DNA from 20 osteosarcoma tumor samples and matched normal tissue in a discovery cohort, as well as 14 samples in a validation cohort. Single-nucleotide variations (SNVs) exhibited a pattern of localized hypermutation called kataegis in 50% of the tumors. We identified p53 pathway lesions in all tumors in the discovery cohort, nine of which were translocations in the first intron of the TP53 gene. Beyond TP53, the RB1, ATRX, and DLG2 genes showed recurrent somatic alterations in 29%-53% of the tumors. These data highlight the power of whole-genome sequencing for identifying recurrent somatic alterations in cancer genomes that may be missed using other methods. Show less

Diabetes mellitus (DM) is a prothrombotic and proinflammatory state. Hyperglycemia (HG) is encountered even in patients without DM. We have shown that combined HG and hyperinsulinemia (HI) in healthy non-diabetic subjects increased circulating tissue factor (TF) and thrombin generation. To understand the changes in platelet and monocyte pathways induced by combined HG and HI in healthy non-diabetic state, we performed whole genome expression profiling of leukocyte-depleted platelets and monocytes before and after 24 hours of combined HG (glucose ~200mg/dL) and HI by glucose infusion clamp in a healthy non-diabetic subject. We defined time-dependent differential mRNA expression (24 versus 0 hour fold change (FC) ≥ 2) common to platelets and monocytes. Ingenuity Pathways Analysis revealed alterations in canonical insulin receptor signaling and coagulation pathways. A preliminary group of 9 differentially expressed genes was selected for qRT-PCR confirmation. Platelet 24 hour sample was compared to the 0 hour sample plus 4 controls. Five transcripts in platelets and 6 in monocytes were confirmed. Platelet GSK3B and PTPN1 were upregulated, and STXBP4 was downregulated in insulin signaling, and F3 and TFPI were upregulated in coagulation pathways. Monocyte, PIK3C3, PTPN11 and TFPI were downregulated. Platelet GSKβ3 and PTPN11 protein and TF antigen in platelets and monocytes was increased. Even in non-diabetic state, HG+HI for 24 hours induces changes in platelets and monocytes. They suggest downregulation of insulin signaling and upregulation of TF. Further studies are needed to elucidate cellular alterations leading to the prothrombotic and proinflammatory state in DM. 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

The specification of the seven retinal cell types from a common pool of retina progenitor cells (RPCs) involves complex interactions between the intrinsic program and the environment. The proneural basic helix-loop-helix (bHLH) transcriptional regulators are key components for the intrinsic programming of RPCs and are essential for the formation of the diverse retinal cell types. However, the extent to which an RPC can re-adjust its inherent program and the mechanisms through which the expression of a particular bHLH factor influences RPC fate is unclear. Previously, we have shown that Neurod1 inserted into the Atoh7 locus activates the retinal ganglion cell (RGC) program in Atoh7-expressing RPCs but not in Neurod1-expressing RPCs, suggesting that Atoh7-expressing RPCs are not able to adopt the cell fate determined by Neurod1, but rather are pre-programmed to produce RGCs. Here, we show that Neurod1-expressing RPCs, which are destined to produce amacrine and photoreceptor cells, can be re-programmed into RGCs when Atoh7 is inserted into the Neurod1 locus. These results suggest that Atoh7 acts dominantly to convert a RPC subpopulation not destined for an RGC fate to adopt that fate. Thus, Atoh7-expressing and Neurod1-expressing RPCs are intrinsically different in their behavior. Additionally, ChIP-Seq analysis identified an Atoh7-dependent enhancer within the intronic region of Nrxn3. The enhancer recognized and used Atoh7 in the developing retina to regulate expression of Nrxn3, but could be forced to use Neurod1 when placed in a different regulatory context. The results indicate that Atoh7 and Neurod1 activate distinct sets of genes in vivo, despite their common DNA-binding element. Show less

Nest breakdown and primordial folliculogenesis of the mouse ovary can be inhibited by progesterone (P4) and Notch signaling inhibitors. However, the relationship between these two signals during this process remains unknown. In the present study, transcript levels of Jagged2, Notch1, and their target, Hey2, increased markedly in ovaries during the beginning stage of folliculogenesis (17.5 days post coitus (dpc) to birth). Maternal P4 levels decreased simultaneously. We found that maternal midpregnancy P4 levels significantly inhibited Jagged2, Notch1, and Hey2 expression, and follicle formation in vitro. Maintaining high maternal P4 levels by daily injection also significantly suppressed the expression of Jagged2, Notch1, and Hey2, and follicle formation during late pregnancy. Based on immunohistochemistry, Jagged2 was localized in oocytes and Notch1 was strongly stained in pre-granulosa cells in 19.5 dpc ovaries. Suppression of their function by antibody addition and RNAi markedly inhibited nest breakdown and follicle formation. Taken together, these results demonstrate that maternal P4 levels during midpregnancy can inhibit the expression of Jagged2 and Notch1, which are involved in primordial folliculogenesis, in the mouse fetal ovary. Show less

To observe the effects of Guanxinkang (GXK) decoction, a compound traditional Chinese herbal medicine, on expressions of peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor α (LXRα) and ATP-binding cassette transporter A1 (ABCA1) in apolipoprotein E (ApoE)-knockout mice with atherosclerosis. Fourteen 6-week-old C57BL/6 J mice were used as normal control group. Seventy 6-week-old ApoE-knockout mice receiving a high-cholesterol diet to induce atherosclerosis were randomly divided into untreated group, simvastatin group and low-dose (concentration of crude drugs at 0.864 g/mL), medium-dose (crude drugs at 1.728 g/mL) and high-dose (crude drugs at 3.456 g/mL) GXK groups. After treated with the drugs for eight weeks continuously, the livers and aortas of mice were separated. The expressions of PPARγ, LXRα and ABCA1 were measured by real-time quantitative polymerase chain reaction and Western blotting respectively. Compared with the normal control group, mRNAs and proteins of PPARγ, LXRα and ABCA1 over-expressed in the untreated group (P<0.05). After the treatment, GXK decoction and simvastatin decreased the expressions of PPARγ, LXRα and ABCA1 (P<0.05). High-dose GXK decoction had more marked effects than low- and medium-dose GXK and simvastatin. The PPARγ-LXRα-ABCA1 pathway is involved in lipid regulation and inflammation activities. Over-expression of the genes has complicated effects on atherosclerosis in ApoE-knockout mice with high-cholesterol diet. GXK decoction has anti-inflammatory and anti-matrix metalloproteinase activities by regulating PPARγ, LXRα and ABCA1 interactions in the ApoE-knockout mice. Show less

This study investigated the impact of chronic exposure to continuous (CoP4) versus cyclic progesterone (CyP4) alone or in combination with 17β-estradiol (E2) on gene expression profiles targeting bioenergetics, metabolism and inflammation in the adult female rat hippocampus. High-throughput qRT-PCR analyses revealed that ovarian hormonal depletion induced by ovariectomy (OVX) led to multiple significant gene expression alterations, which were to a great extent reversed by co-administration of E2 and CyP4. In contrast, co-administration of E2 and CoP4 induced a pattern highly resembling OVX. Bioinformatics analyses further revealed clear disparities in functional profiles associated with E2+CoP4 and E2+CyP4. Genes involved in mitochondrial energy (ATP synthase α subunit; Atp5a1), redox homeostasis (peroxiredoxin 5; Prdx5), insulin signaling (insulin-like growth factor I; Igf1), and cholesterol trafficking (liver X receptor α subtype; Nr1h3), differed in direction of regulation by E2+CoP4 (down-regulation relative to OVX) and E2+CyP4 (up-regulation relative to OVX). In contrast, genes involved in amyloid metabolism (β-secretase; Bace1) differed only in degree of regulation, as both E2+CoP4 and E2+CyP4 induced down-regulation at different efficacy. E2+CyP4-induced changes could be associated with regulation of progesterone receptor membrane component 1(Pgrmc1). In summary, results from this study provide evidence at the molecular level that differing regimens of hormone therapy (HT) can induce disparate gene expression profiles in brain. From a translational perspective, confirmation of these results in a model of natural menopause, would imply that the common regimen of continuous combined HT may have adverse consequences whereas a cyclic combined regimen, which is more physiological, could be an effective strategy to maintain neurological health and function throughout menopausal aging. Show less

The significance of transcription factors PPAR alpha, LXR alpha, and their responsive/target genes for the pathogenesis of atherosclerosis in apolipoprotein E and low-density lipoprotein receptor double deficient (AL) mice fed with high fat and cholesterol (HF) diet were studied. C57BL/6J wild-type (WT) mice were used as control to the AL mice. Plasma lipid metabolites and morphological atherosclerotic lesions in aortic wall were determined. Semi- and real-time quantitative RT-PCR were used to measure gene expression patterns between AL mice and the controls, which were fed with HF or normal chow diet. The results showed that in AL mice fed with HF diet, plasma lipid levels, hepatic lipid accumulation, and atherogenesis together with upregulated PPAR alpha, LXR alpha, and their target genes, i.e., FAT, SCD1, FAS, Angptl3, and apoB100 significantly increased in a 12-week long feeding period. In contrast, apoAI, apoAIV, apoF, LPL, and SR-BI were decreased compared to chow-fed group. In WT mice, PPAR alpha, LXR alpha, FAS, Angpt13, CPT1, apoF, ACOX1, LPL, and SR-BI were increased with HF treatment, while apoAI and apoAIV were decreased markedly. The different changes of lipid metabolism-related genes between AL and WT mice, fed with HF diet or chow diet indicated that the mechanisms of dietary effects on gene mutant mice are different from those of intact WT mice. Since lipid metabolic system defected genetically in AL mice, we suggest that the changes of PPAR alpha, LXR alpha, and their target genes aggravated lipid metabolic disorder in the liver and further accelerated the development of atherosclerosis on a stress of HF diet feeding in AL mice. Show less

Juvenile neuronal ceroid lipofuscinosis is a severe inherited neurodegenerative disease resulting from mutations in CLN3 (ceroid-lipofuscinosis, neuronal 3, juvenile). CLN3 function, and where and when it is expressed during development, is not known. In this study, we generated a knock-in reporter mouse to elucidate CLN3 expression during embryogenesis and after birth and to correlate expression and behavior in a CLN3-deficient mouse. In embryonic brain, expression appeared in the cortical plate. In postnatal brain, expression was prominent in the cortex, subiculum, parasubiculum, granule neurons of the dentate gyrus, and some brainstem nuclei. In adult brain, reporter gene expression waned in most areas but remained in vascular endothelia and the dentate gyrus. Mice homozygous for Cln3 deletion showed two hallmark pathological features of the neuronal ceroid lipofuscinosises: autofluorescent inclusions and lysosomal enzyme elevation. Moreover, CLN3-deficient reporter mice displayed progressive neurological deficits, including impaired motor function, decreased overall activity, acquisition of resting tremors, and increased susceptibility to pentilentetrazole-induced seizures. Notably, seizure induction in heterozygous mice was accompanied by enhanced reporter expression. This model provides us with the unique ability to correlate expression with pathology and behavior, thus facilitating the elucidation of CLN3 function and the pathogenesis of Batten disease. Show less

Human protein kinases make up a large superfamily of homologous proteins, which are related by virtue of their kinase domains (also known as catalytic domains). Here we report the cloning and characterization of a novel human MAST4 (microtubule associated serine/threonine kinase family member 4) gene, which locates on human chromosome 5q13. The MAST4 cDNA is 7587 base pairs in length and encodes a putative protein of 2435 amino acids which contains a serine/threonine kinase domain and a PDZ domain. MAST4 protein has 64%, 63%, 59% and 39% identical aminoacid residues with MAST1, MAST2, MAST3 and MASTL respectively. RT-PCR analysis revealed relatively high expression level of MAST4 in most normal human tissues, with an exception of in testis, small intestine, colon and peripheral blood leukocyte. Show less

Regulators of G-protein signaling (RGS proteins) comprise over 20 different proteins that have been classified into subfamilies on the basis of structural homology. The RZ/A family includes RGSZ2/RGS17 (the most recently discovered member of this family), GAIP/RGS19, RGSZ1/RGS20, and the RGSZ1 variant Ret-RGS. The RGS proteins are GTPase activating proteins (GAPs) that turn off G-proteins and thus negatively regulate the signaling of G-protein coupled receptors (GPCRs). In addition, some RZ/A family RGS proteins are able to modify signaling through interactions with adapter proteins (such as GIPC and GIPN). The RZ/A proteins have a simple structure that includes a conserved amino-terminal cysteine string motif, RGS box and short carboxyl-terminal, which confer GAP activity (RGS box) and the ability to undergo covalent modification and interact with other proteins (amino-terminal). This review focuses on RGS17 and its RZ/A sibling proteins and discusses the similarities and differences among these proteins in terms of their palmitoylation, phosphorylation, intracellular localization and interactions with GPCRs and adapter proteins. The specificity of these RGS protein for different Galpha proteins and receptors, and the consequences for signaling are discussed. The tissue and brain distribution, and the evolving understanding of the roles of this family of RGS proteins in receptor signaling and brain function are highlighted. Show less

Bardet-Biedl syndrome (BBS) is a heterogeneous multisystemic disorder characterized primarily by five cardinal features of retinal degeneration, obesity, polydactyly, hypogenitalism and mental retardation. To date, six distinct BBS loci that have been identified on different chromosomes. BBS4 gene is mapped to 15q22.2-23, which when mutated can cause BBS4. Its protein shows strong homology to O-linked N-acetylglucosamine (O-GlcNAc) transferase. Here we report a splice variant of BBS4, which is 2556 bp in length and has an open reading frame coding a predicted 527 amino-acids protein. RT-PCR shows that the cDNA is widely expressed while it has higher expression levels in pancreas, liver and prostate. Show less

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. Show less