👤 J I Tamura

Malignant phyllodes tumors (MPTs) are rare fibroepithelial breast tumors with no standard treatment for metastatic or recurrent cases. Comprehensive genomic profiling (CGP) has been conducted for MPT; however, its association with treatment remains unclear. A retrospective study was conducted on patients with advanced or recurrent MPTs treated with chemotherapy between 2013 and 2022 at two hospitals, analyzing clinical data, CGP, treatment outcomes, and survival. Five patients with metastatic MPTs who had received chemotherapy were identified. The median age was 55 years (range, 50-66), and all patients were female. As first-line treatment, four patients received doxorubicin plus ifosfamide (AI) combination therapy, while one received doxorubicin monotherapy. Among those treated with AI therapy, the best responses were partial response in three patients and stable disease in one. The median progression-free survival (PFS) for patients treated with AI therapy was 5.3 months. Of the five patients two proceeded to second-line therapy, and one patient received up to fourth-line treatment. Next-generation sequencing-based CGP testing was performed in four cases. One patient with an FGFR1-N546K-mutated MPT achieved a relatively long PFS of 6.8 months with pazopanib therapy, a multi-kinase inhibitor targeting FGFR1 among other kinases, as fourth-line therapy. AI therapy is useful for advanced or recurrent MPTs. The observed clinical benefit of pazopanib in a patient with FGFR1 N546K-mutated MPT suggests that FGFR1 kinase domain mutations may be a relevant factor in responsiveness of FGFR1-targeted therapy. Further data accumulation is warranted. Show less

In the tumor microenvironment, hypoxia and stromal interactions contribute to enhanced malignant behavior in cancer cells. This study aimed to assess whether pancreatic cancer cells with higher malignancy display stronger responses to hypoxia and stromal cells than their less malignant parental cells, and evaluated the underlying mechanisms, focusing on lysophosphatidic acid (LPA) receptor signaling linked to the acquisition of malignant traits. Highly invasive PANC-M10 cells, derived from the parental pancreatic cancer PANC-1 cells, were cultured at 1% O Show less

Lysophosphatidic acid (LPA) receptor-mediated signaling contributes to the pathogenesis of cancer. The tumor microenvironment (TME), composed of cancer cells and surrounding stromal cells, plays a key role in promoting malignant traits, including resistance to anticancer drugs. In this study, we investigated the roles of LPA receptor-1 (LPA Show less

Hypoxia plays a crucial role in driving tumor progression by altering cellular signaling pathways. Lysophosphatidic acid (LPA) receptor signaling regulates malignant properties in cancer cells, including motility and chemoresistance. This study aimed to compare the cellular functions of gastric cancer AGS cells under cobalt chloride (CoCl Show less

In solid tumors, cancer cells adapt to hypoxic and nutrient deprived environments to support malignant progression. This study examined whether hypoxic and low glucose conditions enhance malignant behaviors more strongly in highly migratory MG63-R10 cells, which are derived from osteosarcoma MG-63 cells, compared to parental MG-63 cells, and further investigated whether lysophosphatidic acid (LPA) receptor signaling regulates this adaptation. MG63-R10 and MG-63 cells were cultured under hypoxic (1% O Under 1% O These results suggest that, compared to parental MG-63 cells, highly migratory osteosarcoma MG63-R10 cells adapt their malignant cellular functions to hypoxic and low-glucose conditions through LPA receptor signaling, highlighting this pathway as a potential therapeutic target in aggressive osteosarcomas. The online version contains supplementary material available at 10.1007/s12195-025-00873-y. Show less

The impact of Graves' hyperthyroidism treatment on lipid metabolism remains unclear. This prospective observational study aimed to clarify the changes in lipid profiles and associated metabolic pathways, including cholesterol synthesis, absorption, and low-density lipoprotein (LDL) receptor regulation, following treatment. Seventeen patients newly diagnosed with Graves' hyperthyroidism were enrolled and followed for 6 months after achieving euthyroid status. Serum lipids (total cholesterol, LDL-cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides), apolipoproteins, non-cholesterol sterols (markers of cholesterol synthesis and absorption), proprotein convertase subtilisin/kexin type 9 (PCSK9), and lipoprotein lipase (LPL) levels were measured at baseline, at euthyroid status (Eu-0M), and 6 months after euthyroid status (Eu-6M). After treatment, serum total cholesterol, LDL-C, and HDL-C levels increased rapidly compared to baseline, while triglyceride levels showed a delayed but significant increase at Eu-6M. Levels of apolipoprotein (apo) AI, AII, B, and CIII increased significantly after treatment, whereas apo B-48 increased only at Eu-6M, and apo CII and apo E remained unchanged. Markers of cholesterol synthesis (lathosterol) and absorption (sitosterol, campesterol, and cholestanol) increased significantly after treatment, indicating enhanced cholesterol metabolism. Circulating PCSK9 levels increased significantly and remained elevated, while LPL levels did not change significantly. Treatment of Graves' hyperthyroidism rapidly increases cholesterol levels through enhanced cholesterol synthesis and absorption, possibly mediated by increased circulating PCSK9. Show less

Pancreas-specific lipase and lipase activity measured by immunological assays (Spec cPL and Spec fPL) and colorimetric assays (1,2-o-dilauryl-rac-glycelo-3-glutaric acid-(6'-methylresorufin) ester [DGGR] and triolein), respectively, are used to diagnose pancreatitis in both dogs and cats. However, DGGR and triolein assays may be influenced by extrapancreatic lipases, including hepatic triglyceride lipase (HTGL) and lipoprotein lipase (LPL). To investigate the effect of extrapancreatic lipases on immunological and colorimetric assays by measuring changes in HTGL and LPL activity following heparin administration. Six healthy Beagles and six adult purpose-bred cats were enrolled. HTGL and LPL activities were induced by intravenous heparin administration. Serum samples were collected at baseline and at 5-, 10-, 15-, and 60-min following heparin injection. Spec cPL, Spec fPL, and lipase activities were measured using DGGR and triolein assays, whereas HTGL and LPL activities were measured using their respective assays. Spec cPL and Spec fPL levels showed no significant changes following heparin administration. Conversely, DGGR-based and triolein-based lipase activities, as well as HTGL and LPL activities, were significantly increased after heparin administration in both dogs and cats. HTGL and LPL activities showed significant positive correlations with DGGR-based (P < .001, r = .90 for both) and triolein-based (P < .001, r = .63 and P < .001, r = .68, respectively) lipase activities, but not with Spec cPL and Spec fPL. DGGR- and triolein-based lipase activities are influenced by HTGL and LPL activities, as their substrates are hydrolyzed by pancreatic lipase, HTGL, and LPL. Show less

Endometriosis is hypothesized to result from retrograde menstruation where cell debris including endometrial stromal cells (ESCs) travel through the fallopian tubes. This chronic inflammatory disease is characterized by inflammatory and fibrotic endometrial tissue. We have previously observed reduced expression of the anti-inflammatory factor SERPINA1 in endometriosis-like lesions in a mouse model implanted with human ESCs. Additionally, pro-inflammatory factors present in peritoneal hemorrhage exacerbated inflammation in these grafts, partly through prostaglandin (PG) E2 and thrombin. However, it remains unclear whether the reduction of SERPINA1, in combination with PGE2 and thrombin, synergistically influences the expression of inflammatory factors in endometriosis lesions and the underlying mechanisms. We analyzed RNA sequencing data from ESCs treated with SERPINA1 siRNA and PGE2/thrombin, comparing them to data sets derived from ESCs subjected to either SERPINA1 knockdown or PGE2/thrombin treatment. Comparative analysis identified 49 transcripts that were upregulated under both conditions and enriched for transcription regulatory genes, including SNAI1, HDAC5, PBX1, SOX4, EPAS1, LHX9, and MAFK. Silencing SNAI1, HDAC5, SOX4, EPAS1, or LHX9 suppressed IL6, CXCL8, and IL1B expression, which had been upregulated by SERPINA1 siRNA and PGE2/thrombin. Among these genes, LHX9 expression was significantly elevated in ectopic lesions, predominantly localized to stromal and glandular epithelial cells, with more pronounced expression during the secretory phase. LHX9 levels were also increased in endometriotic lesions compared to the normal endometrium. In conclusion, reduced SERPINA1 expression in ectopic ESCs, combined with PGE2/thrombin, induces inflammatory cytokine expression linked to LHX9. Pharmacological targeting of LHX9 may present a promising therapeutic strategy for mitigating chronic inflammation in endometriotic lesions. Show less

Environmental and genetic factors are suggested to exhibit factor-based association with HDL-cholesterol (HDL-C) levels. However, the population-based effects of environmental and genetic factors have not been compared clearly. We conducted a cross-sectional study using data from the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study to evaluate the population-based impact of smoking, drinking, and genetic factors on low HDL-C. Data from 11,498 men and women aged 35-69 years were collected for a genome-wide association study (GWAS). Sixty-five HDL-C-related SNPs with genome-wide significance (P < 5 × 10 We found that smoking, drinking, daily activity, habitual exercise, egg intake, BMI, age, sex, and the SNPs CETP rs3764261, APOA5 rs662799, LIPC rs1800588, LPL rs328, ABCA1 rs2575876, LIPG rs3786247, and APOE rs429358 were associated with HDL-C levels. The gene-environmental interactions on smoking and drinking were not statistically significant. The PAF for low HDL-C was the highest in men (63.2%) and in rs3764261 (31.5%) of the genetic factors, and the PAFs of smoking and drinking were 23.1% and 41.8%, respectively. The present study showed that the population-based impact of genomic factor CETP rs3764261 for low HDL-C was higher than that of smoking and lower than that of drinking. Show less

Exostosin 1 (EXT1) and exostosin 2 (EXT2)-associated membranous nephropathy (MN) may be associated with active autoimmune disease. We encountered an elderly man who presented with EXT1/EXT2-associated lupus-like MN with full house immune deposits, monoclonal gammopathy of uncertain significance and Sjögren's syndrome. The patient exhibited various other immune abnormalities. Although he did not fulfill the criteria of clinical systemic lupus erythematosus (SLE), he met a stand-alone renal criterion of the Systemic Lupus International Collaborating Clinics (SLICC) 2012. Whether or not a stand-alone renal criterion with EXT1/EXT2 positivity, as in the present patient, can efficiently guide decisions regarding the diagnosis and treatment of SLE remains a clinical dilemma. Show less

RA is an autoimmune disease characterized by destructive polyarthritis. CD4+ T cells are pivotal to its pathogenesis, and our previous study revealed the expression of fibroblast growth factor receptor 1 (FGFR1) is modulated by MTX treatment in CD4+ T cells of RA patients; however, the roles of FGFR1 in CD4+ T cells in the pathogenesis of RA is unclear. Therefore, in this study, we aimed to characterize FGFR1-positive CD4+ T cells in RA patients. The abundance of FGFR1-positive CD4+ T cells in peripheral blood and synovium was determined. Single-cell RNA sequencing (scRNA-seq) was performed on synovial CD4+ T cells to characterize FGFR1-positive cells. In addition, T cell activation status and cytokine production were determined using flow cytometry. The percentage of FGFR1-positive CD4+ T cells in the peripheral blood was higher in RA patients than in healthy controls (P =0.0035). They were also present in the synovium of active RA patients. The results of scRNA-seq revealed that peripheral Th (Tph) cells preferentially expressed FGFR1. Additionally, these FGFR1-positive Tph cells displayed a terminal effector cell phenotype. Consistent with this finding, FGFR1-positive CD4+ T cells in peripheral blood expressed IL-21 and IFN-γ. Our study provides evidence that FGFR1 marks terminal effector Tph cells in patients with RA. Show less

Gout is a common type of acute arthritis that results from elevated serum uric acid (SUA) levels. Recent genome-wide association studies (GWASs) have revealed several novel single nucleotide polymorphism (SNPs) associated with SUA levels. Of these, rs10821905 of A1CF and rs1178977 of BAZ1B showed the greatest and the second greatest significant effect size for increasing SUA level in the Japanese population, but their association with gout is not clear. We examined their association with gout using 1411 clinically-defined Japanese gout patients and 1285 controls, and meta-analyzed our previous gout GWAS data to investigate any association with gout. Replication studies revealed both SNPs to be significantly associated with gout (P = 0.0366, odds ratio [OR] with 95% confidence interval [CI]: 1.30 [1.02-1.68] for rs10821905 of A1CF, P = 6.49 × 10 Show less

A wide range of human diseases result from haploinsufficiency, where the function of one of the two gene copies is lost. Here, we targeted the remaining functional copy of a haploinsufficient gene using CRISPR-mediated activation (CRISPRa) in Show less

Vacuolar protein sorting 9 (VPS9)-ankyrin-repeat protein (Varp) has recently been identified as an effector molecule for two small GTPases-Rab32 and Rab38-in the transport of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes in melanocytes. Although Varp contains a Rab21-guanine nucleotide exchange factor (GEF) domain (i.e., VPS9 domain), since Rab21-GEF activity is not required for Tyrp1 transport, nothing is known about the physiological significance of the Rab21-GEF activity in melanocytes. Here we show by knockdown-rescue experiments that the Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for forskolin-induced dendrite formation of cultured melanocytes. We found that Varp-deficient cells are unable to extend dendrites in response to forskolin stimulation and that reexpression of wild-type Varp or a Rab32/38-binding-deficient mutant Varp(Q509A/Y550A) in Varp-deficient cells completely restores their ability to form dendrites. By contrast, VPS9 mutants (D310A and Y350A) and a vesicle-associated membrane protein 7 (VAMP7)-binding-deficient mutant were unable to support forskolin-induced dendrite formation in Varp-deficient cells. These findings indicate that the Rab21-GEF activity and Rab32/38 binding activity of Varp are required for different melanocyte functions, that is, Rab21 activation by the VPS9 domain is required for dendrite formation, and the Rab32/38 effector function of the ankyrin repeat 1 domain is required for Tyrp1 transport to melanosomes, although VAMP7-binding ability is required for both functions. Show less

The mass spectrometry-based peptidomics approaches have proven its usefulness in several areas such as the discovery of physiologically active peptides or biomarker candidates derived from various biological fluids including blood and cerebrospinal fluid. However, to identify biomarkers that are reproducible and clinically applicable, development of a novel technology, which enables rapid, sensitive, and quantitative analysis using hundreds of clinical specimens, has been eagerly awaited. Here we report an integrative peptidomic approach for identification of lung cancer-specific serum peptide biomarkers. It is based on the one-step effective enrichment of peptidome fractions (molecular weight of 1,000-5,000) with size exclusion chromatography in combination with the precise label-free quantification analysis of nano-LC/MS/MS data set using Expressionist proteome server platform. We applied this method to 92 serum samples well-managed with our SOP (standard operating procedure) (30 healthy controls and 62 lung adenocarcinoma patients), and quantitatively assessed the detected 3,537 peptide signals. Among them, 118 peptides showed significantly altered serum levels between the control and lung cancer groups (p<0.01 and fold change >5.0). Subsequently we identified peptide sequences by MS/MS analysis and further assessed the reproducibility of Expressionist-based quantification results and their diagnostic powers by MRM-based relative-quantification analysis for 96 independently prepared serum samples and found that APOA4 273-283, FIBA 5-16, and LBN 306-313 should be clinically useful biomarkers for both early detection and tumor staging of lung cancer. Our peptidome profiling technology can provide simple, high-throughput, and reliable quantification of a large number of clinical samples, which is applicable for diverse peptidome-targeting biomarker discoveries using any types of biological specimens. Show less

Because Varp (VPS9-ankyrin-repeat protein)/Ankrd27 specifically binds two small GTPases, Rab32 and Rab38, which redundantly regulate the trafficking of melanogenic enzymes in mammalian epidermal melanocytes, it has recently been implicated in the regulation of trafficking of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes. However, the functional interaction between Rab32/38 and Varp and the involvement of the VPS9 domain (i.e. Rab21-GEF domain) in Tyrp1 trafficking have never been elucidated. In this study, we succeeded in identifying critical residues of Rab32/38 and Varp that are critical for the formation of the Rab32/38·Varp complex by performing Ala-based site-directed mutagenesis, and we discovered that a conserved Val residue in the switch II region of Rab32(Val-92) and Rab38(Val-78) is required for Varp binding activity and that its point mutant, Rab38(V78A), does not support Tyrp1 trafficking in Rab32/38-deficient melanocytes. We also identified two critical residues for Rab32/38 binding in the Varp ANKR1 domain and demonstrated that their point mutants, Varp(Q509A) and Varp(Y550A), do not support peripheral melanosomal distribution of Tyrp1 in Varp-deficient cells. Interestingly, the VPS9 domain point mutants, Varp(D310A) and Varp(Y350A), did support Tyrp1 trafficking in Varp-deficient cells, and knockdown of Rab21 had no effect on Tyrp1 distribution. We also found evidence for the functional interaction between a vesicle SNARE VAMP7/TI-VAMP and Varp in Tyrp1 trafficking. These results collectively indicated that both the Rab32/38 binding activity and VAMP7 binding activity of Varp are essential for trafficking of Tyrp1 in melanocytes but that activation of Rab21 by the VPS9 domain is not necessary for Tyrp1 trafficking. Show less

In tetrapod limbs, an anteriormost digit has common traits of small, short and less-phalange morphology. In this study, we focused on three genes, Mkp3, Sef and Tsukushi (TSK), which have anterior-specific or anterior-prominent expression patterns in the developing limb bud at the autopod-forming stage. The anterior expression is not fixed in the period of limb development, but the expression domains of Mkp3, Sef and TSK change considerably from the distal domain to the anterior domain. This change in expression domains, anterior shift, of these genes involves maintenance of gene expression in the anterior side and downregulation in the posterior side. Manipulated overdose of fibroblast growth factor (FGF) in the presumptive digit 2 region of chick forelimb bud results in elongation of cartilage elements of digit 2, suggesting that attenuated FGF signaling, which Mkp3, Sef, and TSK negatively regulate, provides digit 2-specific traits of morphology. The anterior expression of Mkp3 and Sef but not TSK is conserved also in limb buds of the mouse and gecko, and the anterior shift of these genes, accumulation of their transcripts in the anterior side and appropriate regulation of strength of FGF signaling may control species-specific morphology of the anteriormost digit. Show less

HS (heparan sulfate) is synthesized by HS co-polymerases encoded by the EXT1 and EXT2 genes (exostosin 1 and 2), which are known as causative genes for hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumours. It has been thought that the hetero-oligomeric EXT1-EXT2 complex is the biologically relevant form of the polymerase and that targeted deletion of either EXT1 or EXT2 leads to a complete lack of HS synthesis. In the present paper we show, unexpectedly, that two distinct cell lines defective in EXT1 expression indeed produce small but significant amounts of HS chains. The HS chains produced without the aid of EXT1 were shorter than HS chains formed in concert with EXT1 and EXT2. In addition, biosynthesis of HS in EXT1-defective cells was notably blocked by knockdown of either EXT2 or EXTL2 (EXT-like), but not of EXTL3. Then, to examine the roles of EXTL2 in the biosynthesis of HS in EXT1-deficient cells, we focused on the GlcNAc (N-aetylglucosamine) transferase activity of EXTL2, which is involved in the initiation of HS chains by transferring the first GlcNAc to the linkage region. Although EXT2 alone synthesized no heparan polymers on the synthetic linkage region analogue GlcUAbeta1-3Galbeta1-O-C2H4NH-benzyloxycarbonyl, marked polymerization by EXT2 alone was demonstrated on GlcNAcalpha1-4GlcUAbeta1-3Galbeta1-O-C2H4N-benzyloxycarbonyl (where GlcUA is glucuronic acid and Gal is galactose), which was generated by transferring a GlcNAc residue using recombinant EXTL2 on to GlcUAbeta1-3Galbeta1-O-C2H4NH-benzyloxycarbonyl. These findings indicate that the transfer of the first GlcNAc residue to the linkage region by EXTL2 is critically required for the biosynthesis of HS in cells deficient in EXT1. Show less

Patients with ulcerative colitis (UC) have an increased risk for developing colorectal cancer. Because UC tumorigenesis is associated with genomic field defects that can extend throughout the entire colon, including the non-dysplastic mucosa; we hypothesized that the same field defect will include abnormally expressed proteins. Here we applied proteomics to study the protein expression of UC neoplastic progression. The protein profiles of colonic epithelium were compared from 1) UC patients without dysplasia (non-progressors); 2) none-dysplastic colonic tissue from UC patient with high-grade dysplasia or cancer (progressors); 3) high-grade dysplastic tissue from UC progressors and 4) normal colon. We identified protein differential expression associated with UC neoplastic progression. Proteins relating to mitochondria, oxidative activity, calcium-binding proteins were some of interesting classes of these proteins. Network analysis discovered that Sp1 and c-myc proteins may play roles in UC early and late stages of neoplastic progression, respectively. Two over-expressed proteins in the non-dysplastic tissue of UC progressors, CPS1 and S100P, were further confirmed by IHC analysis. Our study provides insight into the molecular events associated with UC neoplastic progression, which could be exploited for the development of protein biomarkers in fields of non-dysplastic mucosa that identify a patient's risk for UC dysplasia. Show less

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes. Show less

The proteins encoded by all of the five cloned human EXT family genes (EXT1, EXT2, EXTL1, EXTL2, and EXTL3), members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the biosynthesis of heparan sulfate. In the Caenorhabditis elegans genome, only two genes, rib-1 and rib-2, homologous to the mammalian EXT genes have been identified. Although rib-2 encodes an N-acetylglucosaminyltransferase involved in initiating the biosynthesis and elongation of heparan sulfate, the involvement of the protein encoded by rib-1 in the biosynthesis of heparan sulfate remains unclear. Here we report that RIB-1 is indispensable for the biosynthesis and for embryonic morphogenesis. Despite little individual glycosyltransferase activity by RIB-1, the polymerization of heparan sulfate chains was demonstrated when RIB-1 was coexpressed with RIB-2 in vitro. In addition, RIB-1 and RIB-2 were demonstrated to interact by pulldown assays. To investigate the functions of RIB-1 in vivo, we depleted the expression of rib-1 by deletion mutagenesis. The null mutant worms showed reduced synthesis of heparan sulfate and embryonic lethality. Notably, the null mutant embryos showed abnormality at the gastrulation cleft formation stage or later and arrested mainly at the 1-fold stage. Nearly 100% of the embryos died before L1 stage, although the differentiation of some of the neurons and muscle cells proceeded normally. Similar phenotypes have been observed in rib-2 null mutant embryos. Thus, RIB-1 in addition to RIB-2 is indispensable for the biosynthesis of heparan sulfate in C. elegans, and the two cooperate to synthesize heparan sulfate in vivo. These findings also show that heparan sulfate is essential for post-gastrulation morphogenic movement of embryonic cells and is indispensable for ensuring the normal spatial organization of differentiated tissues and organs. Show less

Germline mutation and functional loss of EXT1 or EXT2 are commonly found in multiple osteochondromas and predispose to the development of chondrosarcoma. Mutations of EXT1 and EXT2 have rarely been detected in sporadic secondary chondrosarcomas from osteochondroma; these frequently display loss of heterozygosity at the EXT1 and EXT2 loci, but primary chondrosarcomas typically do not. To evaluate promoter methylation (which is an epigenetic gene silencing mechanism) of EXT1 and EXT2, we performed methylation-specific polymerase chain reaction (PCR) for 20 chondrosarcoma cases (12 primary, 3 secondary to osteochondroma, 2 secondary to enchondromatosis, 2 extraskeletal ordinary, and 1 clear cell) and in five cell lines. In addition, mutation analysis of the EXT1 and EXT2 coding regions was performed using PCR-single-strand conformation polymorphism and sequencing analysis for 12 of the 20 chondrosarcoma cases (8 primary, 1 secondary to enchondromatosis, 1 secondary to osteochondroma, and 2 extraskeletal ordinary) and five cell lines. Promoter methylation of EXT1 and EXT2 was not detected in any of the cases, and both EXT1 and EXT2 were expressed in all cell lines. Two missense mutations in EXT2 (D227E and R299H) were detected among the chondrosarcoma cases. When considering tumor development in primary chondrosarcoma, we should include mutations in EXT2, along with the status of other members of the EXT gene family. Show less

Heparan, the common unsulfated precursor of heparan sulfate (HS) and heparin, is synthesized on the glycosaminoglycan-protein linkage region tetrasaccharide GlcUA-Gal-Gal-Xyl attached to the respective core proteins presumably by HS co-polymerases encoded by EXT1 and EXT2, the genetic defects of which result in hereditary multiple exostoses in humans. Although both EXT1 and EXT2 exhibit GlcNAc transferase and GlcUA transferase activities required for the HS synthesis, no HS chain polymerization has been demonstrated in vitro using recombinant enzymes. Here we report in vitro HS polymerization. Recombinant soluble enzymes expressed by co-transfection of EXT1 and EXT2 synthesized heparan polymers with average molecular weights greater than 1.7 x 105 using UDP-[3H]GlcNAc and UDP-GlcUA as donors on the recombinant glypican-1 core protein and also on the synthetic linkage region analog GlcUA-Gal-O-C2H4NH-benzyloxycarbonyl. Moreover, in our in vitro polymerization system, a part time proteoglycan, alpha-thrombomodulin, that is normally modified with chondroitin sulfate served as a polymerization primer for heparan chain. In contrast, no polymerization was achieved with a mixture of individually expressed EXT1 and EXT2 or with acceptor substrates such as N-acetylheparosan oligosaccharides or the linkage region tetrasaccharide-Ser, which are devoid of a hydrophobic aglycon, suggesting the critical requirement of core protein moieties in addition to the interaction between EXT1 and EXT2 for HS polymerization. Show less

The tumor suppressors EXT1 and EXT2 are associated with hereditary multiple exostoses and encode bifunctional glycosyltransferases essential for chain polymerization of heparan sulfate (HS) and its analog, heparin (Hep). Three highly homologous EXT-like genes, EXTL1-EXTL3, have been cloned, and EXTL2 is an alpha1,4-GlcNAc transferase I, the key enzyme that initiates the HS/Hep synthesis. In the present study, truncated forms of EXTL1 and EXTL3, lacking the putative NH2-terminal transmembrane and cytoplasmic domains, were transiently expressed in COS-1 cells and found to harbor alpha-GlcNAc transferase activity. EXTL3 used not only N-acetylheparosan oligosaccharides that represent growing HS chains but also GlcAbeta1-3Galbeta1-O-C2H4NH-benzyloxycarbonyl (Cbz), a synthetic substrate for alpha-GlcNAc transferase I that determines and initiates HS/Hep synthesis. In contrast, EXTL1 used only the former acceptor. Neither EXTL1 nor EXTL3 showed any glucuronyltransferase activity as examined with N-acetylheparosan oligosaccharides. Heparitinase I digestion of each transferase-reaction product showed that GlcNAc had been transferred exclusively through an alpha1,4-configuration. Hence, EXTL3 most likely is involved in both chain initiation and elongation, whereas EXTL1 possibly is involved only in the chain elongation of HS and, maybe, Hep as well. Thus, their acceptor specificities of the five family members are overlapping but distinct from each other, except for EXT1 and EXT2 with the same specificity. It now has been clarified that all of the five cloned human EXT gene family proteins harbor glycosyltransferase activities, which probably contribute to the synthesis of HS and Hep. Show less

The proteins encoded by the EXT1, EXT2, and EXTL2 genes, members of the hereditary multiple exostoses gene family of tumor suppressors, are glycosyltransferases required for the heparan sulfate biosynthesis. Only two homologous genes, rib-1 and rib-2, of the mammalian EXT genes were identified in the Caenorhabditis elegans genome. Although heparan sulfate is found in C. elegans, the involvement of the rib-1 and rib-2 proteins in heparan sulfate biosynthesis remains unclear. In the present study, the substrate specificity of a soluble recombinant form of the rib-2 protein was determined and compared with those of the recombinant forms of the mammalian EXT1, EXT2, and EXTL2 proteins. The present findings revealed that the rib-2 protein was a unique alpha1,4-N-acetylglucosaminyltransferase involved in the biosynthetic initiation and elongation of heparan sulfate. In contrast, the findings confirmed the previous observations that both the EXT1 and EXT2 proteins were heparan sulfate copolymerases with both alpha1,4-N-acetylglucosaminyltransferase and beta1,4-glucuronyltransferase activities, which are involved only in the elongation step of the heparan sulfate chain, and that the EXTL2 protein was an alpha1,4-N-acetylglucosaminyltransferase involved only in the initiation of heparan sulfate synthesis. These findings suggest that the biosynthetic mechanism of heparan sulfate in C. elegans is distinct from that reported for the mammalian system. Show less