👤 Y Yamaguchi

Myocyte disarray of >10% in the heart is broadly accepted as a diagnostic pitfall for hypertrophic cardiomyopathy (HCM) at postmortem. The present study aims to propose an additional diagnostic criterion of HCM. Heart specimens from 1387 serial forensic autopsy cases were examined. Cases with myocyte disarray were extracted and applied to morphometric analysis to determine the amount of myocyte disarray. Comprehensive genetic analysis by using next-generation sequencing was subsequently applied for cases with myocyte disarray. Fifteen cases with myocyte disarray were extracted as candidate cases (1.1%, 11 men and 4 women, aged 48⁻94 years). In terms of the cause of death, only 2 cases were cardiac or possible cardiac death, and the other was non-cardiac death. Six cases showed myocyte disarray of >10% and 3 cases showed myocyte disarray of 5% to 10%. The other 6 cases showed myocyte disarray of <5%. Nine rare variants in 5 HCM-related genes (MYBPC3, MYH7, MYH6, PRKAG2, and CAV3) were found in 8 of 9 cases with myocyte disarray of >5%. The remaining 1 and 6 cases with myocyte disarray of <5% did not have any such variant. Myocyte disarray of >5% with rare variants in related genes might be an appropriate postmortem diagnostic criterion for HCM, in addition to myocyte disarray of 10%. Show less

Mutually exclusive KIT and PDGFRA mutations are considered to be the earliest events in gastrointestinal stromal tumors (GIST), but insufficient for their malignant progression. Herein, we aimed to identify driver genes and signaling pathways relevant to GIST progression. We investigated genetic profiles of 707 driver genes, including mutations, gene fusions, copy number gain or loss, and gene expression for 65 clinical specimens of surgically dissected GIST, consisting of six metastatic tumors and 59 primary tumors from stomach, small intestine, rectum, and esophagus. Genetic alterations included oncogenic mutations and amplification-dependent expression enhancement for oncogenes (OG), and loss of heterozygosity (LOH) and expression reduction for tumor suppressor genes (TSG). We assigned activated OG and inactivated TSG to 27 signaling pathways, the activation of which was compared between malignant GIST (metastasis and high-risk GIST) and less malignant GIST (low- and very low-risk GIST). Integrative molecular profiling indicated that a greater incidence of genetic alterations of driver genes was detected in malignant GIST (96%, 22 of 23) than in less malignant GIST (73%, 24 of 33). Malignant GIST samples groups showed mutations, LOH, and aberrant expression dominantly in driver genes associated with signaling pathways of PI3K (PIK3CA, AKT1, and PTEN) and the cell cycle (RB1, CDK4, and CDKN1B). Additionally, we identified potential PI3K-related genes, the expression of which was upregulated (SNAI1 and TPX2) or downregulated (BANK1) in malignant GIST. Based on our observations, we propose that inhibition of PI3K pathway signals might potentially be an effective therapeutic strategy against malignant progression of GIST. Show less

Heparan sulfate (HS), a linear polysaccharide, is involved in diverse biological functions of various tissues. HS is expressed in pancreatic β-cells and may be involved in β-cell functions. However, the importance of HS for β-cell function remains unknown. Here, we generated mice with β-cell-specific deletion of Ext1 (βExt1CKO), which encodes an enzyme essential for HS synthesis, to investigate the detailed roles of HS in β-cell function. βExt1CKO mice decreased body weights compared with control mice, despite increased food intake. Additionally, βExt1CKO mice showed impaired glucose tolerance associated with decreased insulin secretion upon glucose challenge. Glucose-induced insulin secretion (GIIS) from isolated βExt1CKO islets was also significantly reduced, highlighting the contribution of HS to insulin secretion and glucose homeostasis. The gene expression essential for GIIS was decreased in βExt1CKO islets. Pdx1 and MafA were downregulated in βExt1CKO islets, indicating that HS promoted β-cell development and maturation. BrdU- or Ki67-positive β-cells were reduced in βExt1CKO pancreatic sections, suggesting the involvement of HS in the proliferation of β-cells. Moreover, insufficient vascularization in βExt1CKO islets may contribute to central distribution of α-cells. These data demonstrate HS plays diverse roles in β-cells, and that loss of HS leads to insufficient insulin secretion and dysregulation of glucose homeostasis. Show less

Bone remodeling is a highly coordinated process involving bone formation and resorption, and imbalance of this process results in osteoporosis. It has long been recognized that long-term heparin therapy often causes osteoporosis, suggesting that heparan sulfate (HS), the physiological counterpart of heparin, is somehow involved in bone mass regulation. The role of endogenous HS in adult bone, however, remains unclear. To determine the role of HS in bone homeostasis, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS biosynthesis, in osteoblasts. Resultant conditional mutant mice developed severe osteopenia. Surprisingly, this phenotype is not due to impairment in bone formation but to enhancement of bone resorption. We show that osteoprotegerin (OPG), which is known as a soluble decoy receptor for RANKL, needs to be associated with the osteoblast surface in order to efficiently inhibit RANKL/RANK signaling and that HS serves as a cell surface binding partner for OPG in this context. We also show that bone mineral density is reduced in patients with multiple hereditary exostoses, a genetic bone disorder caused by heterozygous mutations of Ext1, suggesting that the mechanism revealed in this study may be relevant to low bone mass conditions in humans. Show less

Multiple hereditary exostoses (MHE), also known as multiple osteochondromas (MO), is an autosomal dominant disorder characterized by the development of multiple cartilage-capped bone tumors (osteochondromas). The large majority of patients with MHE carry loss-of-function mutations in the EXT1 or EXT2 gene, which encodes a glycosyltransferase essential for heparan sulfate (HS) biosynthesis. Increasing evidence suggests that enhanced bone morphogenetic protein (BMP) signaling resulting from loss of HS expression plays a role in osteochondroma formation in MHE. Palovarotene (PVO) is a retinoic acid receptor γ selective agonist, which is being investigated as a potential drug for fibrodysplasia ossificans progressiva (FOP), another genetic bone disorder with features that overlap with those of MHE. Here we show that PVO inhibits osteochondroma formation in the Fsp1 Show less

Multiple hereditary exostoses (MHE) is characterized by the development of numerous benign bony tumors (osteochondromas). Although it has been well established that MHE is caused by mutations in EXT1 and EXT2, which encode glycosyltransferase essential for heparan sulfate (HS) biosynthesis, the cellular origin and molecular mechanisms of MHE remain elusive. Here, we show that in Ext1 mutant mice, osteochondromas develop from mesenchymal stem cell-like progenitor cells residing in the perichondrium, and we show that enhanced BMP signaling in these cells is the primary signaling defect that leads to osteochondromagenesis. We demonstrate that progenitor cells in the perichondrium, including those in the groove of Ranvier, highly express HS and that Ext1 ablation targeted to the perichondrium results in the development of osteochondromas. Ext1-deficient perichondrial progenitor cells show enhanced BMP signaling and increased chondrogenic differentiation both in vitro and in vivo. Consistent with the functional role for enhanced BMP signaling in osteochondromagenesis, administration of the small molecule BMP inhibitor LDN-193189 suppresses osteochondroma formation in two MHE mouse models. Together, our results demonstrate a role for enhanced perichondrial BMP signaling in osteochondromagenesis in mice, and they suggest the possibility of pharmacological treatment of MHE with BMP inhibitors. Show less

Here, we explored the genetic interactions between diabetes and oncogenic single-nucleotide polymorphisms (SNPs) that determine colorectal cancer (CRC) morbidity. 8q24 rs6983267 polymorphism analysis and cDNA microarray were performed in 107 CRCs to identify the genes associated with diabetes and the oncogenic SNP. Then clinical significance of the gene was validated in 132 CRCs. Meta-analysis of microarray data and diabetic comorbidity was performed. Of genes associated with a minor SNP allele at 8q24, diabetes, and MYC overexpression, apolipoprotein A-IV (ApoA-IV) was associated with oncogenesis and poor prognosis in CRC patients. Patients with high ApoA-IV expression showed significantly poorer prognosis by univariate and multivariate analysis. Meta-analysis revealed lipid metabolism was associated with ApoA-IV-related oncogenesis in diabetic patients. Changes in lipid metabolism associated with aberrant expression of ApoA-IV were risks for CRC oncogenesis. Show less

The hepatocyte paraffin 1 (Hep Par 1) antibody is widely used as a hepatocyte marker, recognizing carbamoyl phosphate synthetase 1 (CPS1), an essential component of the urea cycle. Various missense, nonsense, and frameshift mutations occur in the CPS1 gene. In neonatal patients with homozygous CPS1 deficiency (CPS1D), urea cycle defects with resulting severe hyperammonemia can be fatal, though liver transplantation provides a complete cure for CPS1D. We performed Hep Par 1 immunostaining in the explanted livers of 10 liver transplant patients with CPS1D. Seven were negative for Hep Par 1 in the hepatocytes and the other three showed normal diffuse granular cytoplasmic staining. As expected, all three Hep Par 1-positive patients had at least one missense mutation, and all four patients who had only nonsense or frameshift mutations were Hep Par 1-negative. The other three patients were unexpectedly negative for Hep Par 1, even though each had one missense mutation. These results suggest that CPS1D can be related to the loss of Hep Par 1 reactivity due to the loss of protein production, a one amino acid substitution resulting in an abortive protein product, or both. Hep Par 1 immunohistochemistry can be used as a simple method to confirm CPS1D. Show less

Proteoglycans (PGs), a family of glycosaminoglycan (GAG)-protein glycoconjugates, contribute to animal physiology through interactions between their glycan chains and growth factors, chemokines and adhesion molecules. However, it remains unclear how GAG structures are changed during the aging process. Here, we found that polyamine levels are correlated with the expression level of heparan sulfate (HS) in human skin. In cultured cell lines, the EXT1 and EXT2 enzymes, initiating HS biosynthesis, were stimulated at the translational level by polyamines. Interestingly, the initiation codon recognition by 43S preinitiation complex during EXT2 translation is suppressed by let-7b, a member of the let-7 microRNA family, through binding at the N-terminal amino acid coding sequence in EXT2 mRNA. Let-7b-mediated suppression of initiation codon depends on the length of 5'-UTR of EXT2 mRNA and its suppression is inhibited in the presence of polyamines. These findings provide new insights into the HS biosynthesis related to miRNA and polyamines. Show less

Accumulation of amyloid-β (Aβ) peptide in the brain is the first critical step in the pathogenesis of Alzheimer's disease (AD). Studies in humans suggest that Aβ clearance from the brain is frequently impaired in late-onset AD. Aβ accumulation leads to the formation of Aβ aggregates, which injure synapses and contribute to eventual neurodegeneration. Cell surface heparan sulfates (HSs), expressed on all cell types including neurons, have been implicated in several features in the pathogenesis of AD including its colocalization with amyloid plaques and modulatory role in Aβ aggregation. We show that removal of neuronal HS by conditional deletion of the Ext1 gene, which encodes an essential glycosyltransferase for HS biosynthesis, in postnatal neurons of amyloid model APP/PS1 mice led to a reduction in both Aβ oligomerization and the deposition of amyloid plaques. In vivo microdialysis experiments also detected an accelerated rate of Aβ clearance in the brain interstitial fluid, suggesting that neuronal HS either inhibited or represented an inefficient pathway for Aβ clearance. We found that the amounts of various HS proteoglycans (HSPGs) were increased in postmortem human brain tissues from AD patients, suggesting that this pathway may contribute directly to amyloid pathogenesis. Our findings have implications for AD pathogenesis and provide insight into therapeutic interventions targeting Aβ-HSPG interactions. Show less

Adeno-associated virus 2 (AAV2) and adenovirus 5 (Ad5) are promising gene therapy vectors. Both display liver tropism and are currently thought to enter hepatocytes in vivo through cell surface heparan sulfate proteoglycans (HSPGs). To test directly this hypothesis, we created mice that lack Ext1, an enzyme required for heparan sulfate biosynthesis, in hepatocytes. Ext1(HEP) mutant mice exhibit an 8-fold reduction of heparan sulfate in primary hepatocytes and a 5-fold reduction of heparan sulfate in whole liver tissue. Conditional hepatocyte Ext1 gene deletion greatly reduced AAV2 liver transduction following intravenous injection. Ad5 transduction requires blood coagulation factor X (FX); FX binds to the Ad5 capsid hexon protein and bridges the virus to HSPGs on the cell surface. Ad5.FX transduction was abrogated in primary hepatocytes from Ext1(HEP) mice. However, in contrast to the case with AAV2, Ad5 transduction was not significantly reduced in the livers of Ext1(HEP) mice. FX remained essential for Ad5 transduction in vivo in Ext1(HEP) mice. We conclude that while AAV2 requires HSPGs for entry into mouse hepatocytes, HSPGs are dispensable for Ad5 hepatocyte transduction in vivo. This study reopens the question of how adenovirus enters cells in vivo. Our understanding of how viruses enter cells, and how they can be used as therapeutic vectors to manage disease, begins with identification of the cell surface receptors to which viruses bind and which mediate viral entry. Both adeno-associated virus 2 and adenovirus 5 are currently thought to enter hepatocytes in vivo through heparan sulfate proteoglycans (HSPGs). However, direct evidence for these conclusions is lacking. Experiments presented herein, in which hepatic heparan sulfate synthesis was genetically abolished, demonstrated that HSPGs are not likely to function as hepatocyte Ad5 receptors in vivo. The data also demonstrate that HSPGs are required for hepatocyte transduction by AAV2. These results reopen the question of the identity of the Ad5 receptor in vivo and emphasize the necessity of demonstrating the nature of the receptor by genetic means, both for understanding Ad5 entry into cells in vivo and for optimization of Ad5 vectors as therapeutic agents. Show less

Although many single nucleotide polymorphisms (SNPs) have been identified to be associated with metabolic syndrome (MetS), there was only a slight improvement in the ability to predict future MetS by the simply addition of SNPs to clinical risk markers. To improve the ability to predict future MetS, combinational effects, such as SNP-SNP interaction, SNP-environment interaction, and SNP-clinical parameter (SNP × CP) interaction should be also considered. We performed a case-control study to explore novel SNP × CP interactions as risk markers for MetS based on health check-up data of Japanese male employees. We selected 99 SNPs that were previously reported to be associated with MetS and components of MetS; subsequently, we genotyped these SNPs from 360 cases and 1983 control subjects. First, we performed logistic regression analyses to assess the association of each SNP with MetS. Of these SNPs, five SNPs were significantly associated with MetS (P < 0.05): LRP2 rs2544390, rs1800592 between UCP1 and TBC1D9, APOA5 rs662799, VWF rs7965413, and rs1411766 between MYO16 and IRS2. Furthermore, we performed multiple logistic regression analyses, including an SNP term, a CP term, and an SNP × CP interaction term for each CP and SNP that was significantly associated with MetS. We identified a novel SNP × CP interaction between rs7965413 and platelet count that was significantly associated with MetS [SNP term: odds ratio (OR) = 0.78, P = 0.004; SNP × CP interaction term: OR = 1.33, P = 0.001]. This association of the SNP × CP interaction with MetS remained nominally significant in multiple logistic regression analysis after adjustment for either the number of MetS components or MetS components excluding obesity. Our results reveal new insight into platelet count as a risk marker for MetS. Show less

Heparan sulfate (HS) is a highly modified glycosaminoglycan (GAG) bound to a core protein to form heparan sulfate proteoglycans (HSPGs) that are vital in many cellular processes ranging from development to adult physiology, as well as in disease, through interactions with various protein ligands. This study aimed to elucidate the role of HS in corneal epithelial homeostasis and wound healing. An inducible quadruple transgenic mouse model was generated to excise Ext1 and Ndst1, which encode the critical HS chain elongation enzyme and N-deacetylase/N-sulfotransferase, respectively, in keratin 14-positive cells upon doxycycline induction. EXT(Δ/ΔCEpi) mice (deletion of Ext1 in corneal epithelium) induced at P20 presented progressive thinning of the corneal epithelium with a significant loss in the number of epithelial layers by P55. EXT(Δ/ΔCEpi) mice presented tight junction disruption, loss of cell-basement membrane adhesion complexes, and impaired wound healing. Interestingly, EXT(Δ/ΔCEpi) and NDST(Δ/ΔCEpi) mice presented an increase in cell proliferation, which was assayed by both Ki67 staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Moreover, EXT(Δ/ΔCEpi) mice presented compromised epithelial stratification 7 days after a debridement wound. The conditional knockout of HS from keratocytes using the keratocan promoter led to no corneal abnormalities or any disruption in wound healing. Corneal epithelial cells require HS for maintaining corneal homeostasis, and the loss of epithelial HS leads to both impaired wound healing and impaired corneal stratification. Show less

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. Show less

Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries, with peak incidence near the time of puberty. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3-4:1 overall, but is even higher for tumours located in the pineal region. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway. Show less

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism. Show less

Long bones are integral components of the limb skeleton. Recent studies have indicated that embryonic long bone development is altered by mutations in Ext genes and consequent heparan sulfate (HS) deficiency, possibly due to changes in activity and distribution of HS-binding/growth plate-associated signaling proteins. Here we asked whether Ext function is continuously required after birth to sustain growth plate function and long bone growth and organization. Compound transgenic Ext1(f/f);Col2CreERT mice were injected with tamoxifen at postnatal day 5 (P5) to ablate Ext1 in cartilage and monitored over time. The Ext1-deficient mice exhibited growth retardation already by 2weeks post-injection, as did their long bones. Mutant growth plates displayed a severe disorganization of chondrocyte columnar organization, a shortened hypertrophic zone with low expression of collagen X and MMP-13, and reduced primary spongiosa accompanied, however, by increased numbers of TRAP-positive osteoclasts at the chondro-osseous border. The mutant epiphyses were abnormal as well. Formation of a secondary ossification center was significantly delayed but interestingly, hypertrophic-like chondrocytes emerged within articular cartilage, similar to those often seen in osteoarthritic joints. Indeed, the cells displayed a large size and round shape, expressed collagen X and MMP-13 and were surrounded by an abundant Perlecan-rich pericellular matrix not seen in control articular chondrocytes. In addition, ectopic cartilaginous outgrowths developed on the lateral side of mutant growth plates over time that resembled exostotic characteristic of children with Hereditary Multiple Exostoses, a syndrome caused by Ext mutations and HS deficiency. In sum, the data do show that Ext1 is continuously required for postnatal growth and organization of long bones as well as their adjacent joints. Ext1 deficiency elicits defects that can occur in human skeletal conditions including trabecular bone loss, osteoarthritis and HME. Show less

Lymphocyte homing to peripheral lymph nodes (PLNs) is mediated by multistep interactions between lymphocytes and high endothelial venules (HEVs). Heparan sulfate (HS) has been implicated in the presentation of chemokines on the surface of HEVs during this process. However, it remains unclear whether this cell surface presentation is a prerequisite for lymphocyte homing. In this study, we generated conditional knockout (cKO) mice lacking Ext1, which encodes a glycosyltransferase essential for HS synthesis, by crossing Ext1(flox/flox) mice with GlcNAc6ST-2-Cre transgenic mice expressing Cre recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated in PLN HEVs but retained in other blood vessels in the cKO mice. The accumulation of a major secondary lymphoid tissue chemokine, CCL21, on HEVs was also abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated that lymphocyte homing to PLNs was diminished in the cKO mice by 30-40%. Consistent with this result, contact hypersensitivity responses were also diminished in the cKO mice. The residual lymphocyte homing to PLNs in the cKO mice was dependent on pertussis toxin-sensitive Gi protein signaling, in which lysophosphatidic acid-mediated signaling was partly involved. These results suggest that chemokine presentation by HS on the surface of HEVs facilitates but is not absolutely required for lymphocyte homing. Show less

During limb skeletogenesis the cartilaginous long bone anlagen and their growth plates become delimited by perichondrium with which they interact functionally. Yet, little is known about how, despite being so intimately associated with cartilage, perichondrium acquires and maintains its distinct phenotype and exerts its border function. Because perichondrium becomes deranged and interrupted by cartilaginous outgrowths in Hereditary Multiple Exostoses (HME), a pediatric disorder caused by EXT mutations and consequent heparan sulfate (HS) deficiency, we asked whether EXT genes and HS normally have roles in establishing its phenotype and function. Indeed, conditional Ext1 ablation in perichondrium and lateral chondrocytes flanking the epiphyseal region of mouse embryo long bone anlagen - a region encompassing the groove of Ranvier - caused ectopic cartilage formation. A similar response was observed when HS function was disrupted in long bone anlagen explants by genetic, pharmacological or enzymatic means, a response preceded by ectopic BMP signaling within perichondrium. These treatments also triggered excess chondrogenesis and cartilage nodule formation and overexpression of chondrogenic and matrix genes in limb bud mesenchymal cells in micromass culture. Interestingly, the treatments disrupted the peripheral definition and border of the cartilage nodules in such a way that many nodules overgrew and fused with each other into large amorphous cartilaginous masses. Interference with HS function reduced the physical association and interactions of BMP2 with HS and increased the cell responsiveness to endogenous and exogenous BMP proteins. In sum, Ext genes and HS are needed to establish and maintain perichondrium's phenotype and border function, restrain pro-chondrogenic signaling proteins including BMPs, and restrict chondrogenesis. Alterations in these mechanisms may contribute to exostosis formation in HME, particularly at the expense of regions rich in progenitor cells including the groove of Ranvier. Show less

Multidrug resistance protein 2 (MRP2, ABCC2) is localized to the apical membrane of hepatocytes and played an important role in the biliary excretion of a broad range of endogenous and xenobiotic compounds and drugs, such as pravastatin. However, the effects of statins on MRP2 in the liver and the precise mechanisms of their actions have been obscure. The goal of this study was to determine the regulatory molecular mechanism for statin-induced MRP2 expression in hepatocytes. In vitro and in vivo studies suggested that pitavastatin increased MRP2 expression. Pitavastatin promoted liver X receptor (LXR) α/β translocation from the cytosol to nuclei, resulting in LXR activation. Deletion and mutational analysis suggested that the potential sterol regulatory element (SRE) played a major role in the observed modulation of MRP2 expression by pitavastatin. Furthermore pitavastatin increased the protein-DNA complex, and when SRE was mutated, stimulation of the protein-DNA complex by pitavastatin was decreased. It was demonstrated that pitavastatin upregulated MRP2 expression by an SREBP regulatory pathway in hepatocytes and that the actions of statins may lead to improve the biliary excretion of MRP2 substrates. Show less

In Japanese populations, we performed a replication study of genetic loci previously identified in European-descent populations as being associated with lipid levels and risk of coronary artery disease (CAD). We genotyped 48 single nucleotide polymorphisms (SNPs) from 22 candidate loci that had previously been identified by genome-wide association (GWA) meta-analyses for low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and/or triglycerides in Europeans. We selected 22 loci with 2 parallel tracks from 95 reported loci: 16 significant loci (p<1 × 10(-30) in Europeans) and 6 other loci including those with suggestive evidence of lipid associations in 1292 GWA-scanned Japanese samples. Genotyping was done in 4990 general population samples, and 1347 CAD cases and 1337 controls. For 9 SNPs, we further examined CAD associations in an additional panel of 3052 CAD cases and 6335 controls. Significant lipid associations (one-tailed p<0.05) were replicated for 18 of 22 loci in Japanese samples, with significant inter-ethnic heterogeneity at 4 loci-APOB, APOE-C1, CETP, and APOA5-and allelic heterogeneity. The strongest association was detected at APOE rs7412 for LDL-C (p=1.3 × 10(-41)), CETP rs3764261 for HDL-C (p=5.2 × 10(-24)), and APOA5 rs662799 for triglycerides (p=5.8 × 10(-54)). CAD association was replicated and/or verified for 4 loci: SORT1 rs611917 (p=1.7 × 10(-8)), APOA5 rs662799 (p=0.0014), LDLR rs1433099 (p=2.1 × 10(-7)), and APOE rs7412 (p=6.1 × 10(-13)). Our results confirm that most of the tested lipid loci are associated with lipid traits in the Japanese, further indicating that in genetic susceptibility to lipid levels and CAD, the related metabolic pathways are largely common across the populations, while causal variants at individual loci can be population-specific. Show less

Heparan sulfate (HS) has been implicated in regulating cell fate decisions during differentiation of embryonic stem cells (ESCs) into advanced cell types. However, the necessity and the underlying molecular mechanisms of HS in early cell lineage differentiation are still largely unknown. In this study, we examined the potential of EXT1(-/-) mouse ESCs (mESCs), that are deficient in HS, to differentiate into primary germ layer cells. We observed that EXT1(-/-) mESCs lost their differentiation competence and failed to differentiate into Pax6(+)-neural precursor cells and mesodermal cells. More detailed analyses highlighted the importance of HS for the induction of Brachyury(+) pan-mesoderm as well as normal gene expression associated with the dorso-ventral patterning of mesoderm. Examination of developmental cell signaling revealed that EXT1 ablation diminished FGF and BMP but not Wnt signaling. Furthermore, restoration of FGF and BMP signaling each partially rescued mesoderm differentiation defects. We further show that BMP4 is more prone to degradation in EXT1(-/-) mESCs culture medium compared with that of wild type cells. Therefore, our data reveal that HS stabilizes BMP ligand and thereby maintains the BMP signaling output required for normal mesoderm differentiation. In summary, our study demonstrates that HS is required for ESC pluripotency, in particular lineage specification into mesoderm through facilitation of FGF and BMP signaling. Show less

Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism. Show less

In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number. Show less

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance. Show less

Specific interactions of growth factors with heparan sulfate may function as "switches" to regulate stages of branching morphogenesis in developing mammalian organs, such as breast, lung, salivary gland and kidney, but the evidence derives mostly from studies of explanted tissues or cell culture (Shah et al., 2004). We recently provided in vivo evidence that inactivation of Ndst1, the predominant N-deacetylase/N-sulfotransferase gene essential for the formation of mature heparan sulfate, results in a highly specific defect in murine lobuloalveolar development (Crawford et al., 2010). Here, we demonstrate a highly penetrant dramatic defect in primary branching by mammary epithelial-specific inactivation of Ext1, a subunit of the copolymerase complex that catalyzes the formation of the heparan sulfate chain. In contrast to Ext1 deletion, inactivation of Hs2st (which encodes an enzyme required for 2-O-sulfation of uronic acids in heparan sulfate) did not inhibit ductal formation but displayed markedly decreased secondary and ductal side-branches as well as fewer bifurcated terminal end buds. Targeted conditional deletion of c-Met, the receptor for HGF, in mammary epithelial cells showed similar defects in secondary and ductal side-branching, but did not result in any apparent defect in bifurcation of terminal end buds. Although there is published evidence indicating a role for 2-O sulfation in HGF binding, primary epithelial cells isolated from Hs2st conditional deletions were able to activate Erk in the presence of HGF and there appeared to be only a slight reduction in HGF-mediated c-Met phosphorylation in these cells compared to control. Thus, both c-Met and Hs2st play important, but partly independent, roles in secondary and ductal side-branching. When considered together with previous studies of Ndst1-deficient glands, the data presented here raise the possibility of partially-independent regulation by heparan sulfate-dependent pathways of primary ductal branching, terminal end bud bifurcation, secondary branching, ductal side-branching and lobuloalveolar formation. Show less

Multiple Hereditary Exostoses (MHE) syndrome is caused by haploinsufficiency in Golgi-associated heparan sulfate polymerases EXT1 or EXT2 and is characterized by formation of exostoses next to growing long bones and other skeletal elements. Recent mouse studies have indicated that formation of stereotypic exostoses requires a complete loss of Ext expression, suggesting that a similar local loss of EXT function may underlie exostosis formation in patients. To further test this possibility and gain greater insights into pathogenic mechanisms, we created heterozygous Ext1(+/-) and compound Ext1(+/-)/Ext2(+/-) mice. Like Ext2(+/-) mice described previously (Stickens et al. Development 132:5055), Ext1(+/-) mice displayed rib-associated exostosis-like outgrowths only. However, compound heterozygous mice had nearly twice as many outgrowths and, more importantly, displayed stereotypic growth plate-like exostoses along their long bones. Ext1(+/-)Ext2(+/-) exostoses contained very low levels of immuno-detectable heparan sulfate, and Ext1(+/-)Ext2(+/-) chondrocytes, endothelial cells and fibroblasts in vitro produced shortened heparan sulfate chains compared to controls and responded less vigorously to exogenous factors such as FGF-18. We also found that rib outgrowths formed in Ext1(f/+)Col2Cre and Ext1(f/+)Dermo1Cre mice, suggesting that ectopic skeletal tissue can be induced by conditional Ext ablation in local chondrogenic and/or perichondrial cells. The study indicates that formation of stereotypic exostoses requires a significant, but not complete, loss of Ext expression and that exostosis incidence and phenotype are intimately sensitive to, and inversely related to, Ext expression. The data also indicate that the nature and organization of ectopic tissue may be influenced by site-specific anatomical cues and mechanisms. Show less

Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1(f/f) and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1(f/f) mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1(f/f) mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-catenin(f/f) mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth. Show less

In populations of East Asian descent, we performed a replication study of loci previously identified in populations of European descent as being associated with obesity measures such as BMI and type 2 diabetes. We genotyped 14 single nucleotide polymorphisms (SNPs) from 13 candidate loci that had previously been identified by genome-wide association meta-analyses for obesity measures in Europeans. Genotyping was done in 18,264 participants from two general Japanese populations. For SNPs showing an obesity association in Japanese individuals, we further examined diabetes associations in up to 6,781 cases and 7,307 controls from a subset of the original, as well as from additional populations. Significant obesity associations (p < 0.1 two-tailed, concordant direction with previous reports) were replicated for 11 SNPs from the following ten loci in Japanese participants: SEC16B, TMEM18, GNPDA2, BDNF, MTCH2, BCDIN3D-FAIM2, SH2B1-ATP2A1, FTO, MC4R and KCTD15. The strongest effect was observed at TMEM18 rs4854344 (p = 7.1 × 10(-7) for BMI). Among the 11 SNPs showing significant obesity association, six were also associated with diabetes (OR 1.05-1.17; p = 0.04-2.4 × 10(-7)) after adjustment for BMI in the Japanese. When meta-analysed with data from the previous reports, the BMI-adjusted diabetes association was found to be highly significant for the FTO locus in East Asians (OR 1.13; 95% CI 1.09-1.18; p = 7.8 × 10(-10)) with substantial inter-ethnic heterogeneity (p = 0.003). We confirmed that ten candidate loci are associated with obesity measures in the general Japanese populations. Six (of ten) loci exert diabetogenic effects in the Japanese, although relatively modest in size, and independently of increased adiposity. Show less