Akira Katsumi

Human ryudocan from endothelium-like cells binds basic fibroblast growth factor, midkine, and tissue factor pathway inhibitor

Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

The PAR complex targets Tiam1 to adhesions, where it interacts with talin to promote adhesion-induced Rac1 activation, cell spreading, and migration.

Genetic analysis of protein C deficiency in nineteen Japanese families : Five recurrent defects can explain half of the deficiencies

We have been studying the molecular basis of protein C deficiency. In this study, we determined the molecular defects of protein C deficiency in 19 Japanese families by using a strategy combining polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) analysis. We identified 10 missense mutations, 1 in-frame deletion, 1 frameshift deletion, 1 frameshift addition, and 1 splice site mutation, 5 of which were novel. From the results of genetic analysis of 67 Japanese families with protein C deficiency reported in this and previous studies, the recurrent defects including Phe139Val and Met365Ile substitutions and a Lys150 d letion, a G8857 deletion, and a splice site mutation of G3079A were only found in Japanese subjects and seemed to be a founder effect. In contrast, Arg169Trp, Arg286His, Val297Met, and Asp359Asn substitutions, all occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These 9 recurrent molecular defects were found in 43 families in total, accounting 64% of Japanese families with protein C deficiency. In particular, the recurrent defects of Phe139Val, Arg169Trp, Va1297Met, and Met36-4Ile substitutions and a G8857 deletion were found in 33 families in total, accounting for 49% of Japanese families with protein C deficiency. For the identification of the genetic defect in Japanese patients with protein C deficiency, screening of these recurrent defects by using restriction enzyme cleavage is a rational method.

BCR-ABL-independent and RAS / MAPK pathway-dependent form of imatinib resistance in Ph-positive acute lymphoblastic leukemia cell line with activation of EphB4.

Objective: We investigated the mechanism responsible for imatinib (IM) resistance in Philadelphia chromosome‐positive acute lymphoblastic leukemia (Ph+ ALL) cell lines. Methods: We established cell lines from a patient with Ph+ ALL at the time of first diagnosis and relapsed phase and designated as NPhA1 and NPhA2, respectively. We also derived IM‐resistant cells, NPhA2/STIR, from NPhA2 under gradually increasing IM concentrations. Results: NPhA1 was sensitive to IM (IC50 0.05 μm) and NPhA2 showed mild IM resistance (IC50 0.3 μm). NPhA2/STIR could be maintained in the presence of 10 μm IM. Phosphorylation of MEK and ERK was slightly elevated in NPhA2 and significantly elevated in NPhA2/STIR compared to NPhA1 cells. After treatment with IM, phosphorylation of MEK and ERK was not suppressed but rather increased in NPhA2 and NPhA2/STIR. Active RAS was also increased markedly in NPhA2/STIR after IM treatment. The expression of BCL‐2 was increased in NPhA2 compared to NPhA1, but no further increase in NPhA2/STIR. Proliferation of NPhA2/STIR was significantly inhibited by a combination of MEK inhibitor and IM. Analysis of tyrosine phosphorylation status with a protein tyrosine kinase array showed increased phosphorylation of EphB4 in NPhA2/STIR after IM treatment. Although transcription of EphB4 was suppressed in NPhA1 and NPhA2 after IM treatment, it was not suppressed and its ligand, ephrinB2, was increased in NPhA2/STIR. Suppression of EphB4 transcripts by introducing short hairpin RNA into NPhA2/STIR partially restored their sensitivity to IM. Conclusions: These results suggest a new mechanism of IM resistance mediated by the activation of RAS/MAPK pathway and EphB4.

Donor single nucleotide polymorphism in the CCR9 gene affects the incidence of skin GVHD.

The interactions between chemokines and their receptors may have an important role in initiating GVHD after allogeneic hematopoietic SCT (allo-HSCT). CCL25 and CCR9 are unique because they are exclusively expressed in epithelial cells and in Peyers patches of the small intestine. We focused on rs12721497 (G926A), one of the non-synonymous single nucleotide polymorphisms (SNPs) in the CCR9 gene, and analyzed the SNP of donors in 167 consecutive patients who received allo-HSCT from an HLA-identical sibling donor. Genotypes were tested for associations with acute and chronic GVHD in each organ and transplant outcome. Multivariate analyses showed that the genotype 926AG was significantly associated with the incidence of acute stage ⩾2 skin GVHD (hazard ratio: 3.2; 95% confidence interval (95% CI): 1.1–9.1; P=0.032) and chronic skin GVHD (hazard ratio: 4.1; 95% CI: 1.1–15; P=0.036), but not with GVHD in other organs or with relapse, non-relapse mortality or OS. To clarify the functional differences between genotypes, each SNP in retroviral vectors was transfected into Jurkat cells. In chemotaxis assays, the 926G transfectant showed greater response to CCL25 than the 926A transfectant. In conclusion, more active homing of CCR9-926AG T cells to Peyers patches may produce changes in Ag presentation and result in increased incidence of skin GVHD.

LRP16 is fused to RUNX1 in monocytic leukemia cell line with t(11;21)(q13;q22).

Objective: The RUNX1 (also known as AML1) gene is observed frequently as the target of chromosomal rearrangements in human acute leukemia. We describe here a previously unreported rearrangement, t(11;21)(q13;q22), that disrupts the RUNX1 gene in a patient with acute leukemia and the molecular analysis of the fusion gene.Methods: We have established a monocytic leukemia cell line, ELAM‐1, from a patient with acute leukemia evolving from myelodysplastic syndrome (MDS). Translocation (11;21) (q13;q22) was observed in both patient leukemia cells and ELAM‐1.Results: The split signal of RUNX1 was detected by fluorescence in situ hybridization and indicated the involvement of RUNX1 in ELAM‐1. Using 3′‐ Rapid amplification of cDNA ends and reverse transcription‐Polymerase chain reaction analysis, we detected both RUNX1 (exon 5)‐LRP16 and RUNX1 (exon 6)‐LRP16 transcripts, suggesting that the RUNX1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. Reciprocal LRP16‐RUNX1 fusion was also detected.Conclusions: We identified a novel RUNX1 fusion partner, LRP16 on 11q13 involving t(11;21)(q13;q22). Although it was reported that overexpression of LRP16 promotes human breast cancer cell proliferation, the function of LRP16 in leukemia remains to be studied. This fusion gene and cell line may provide a new research tool to investigate the mechanism of leukemogenesis generated by the RUNX1 fusion gene.

Y654 of β-catenin is essential for FLT3/ITD-related tyrosine phosphorylation and nuclear localization of β-catenin.

β‐Catenin plays a dual role as a key effecter in the regulation of adherens junctions as well as a transcriptional co‐activator. Tyrosine phosphorylation of β‐catenin affects the cell adhesion, migration, and gene transcription in many types of human cancer cells, including acute myeloid leukemia cells with FLT3 internal tandem duplication (FLT3/ITD‐AML). Here, we investigated the relationship between three tyrosine residues (Y86, Y142, and Y654) in β‐catenin and oncogenic FLT3/ITD kinase. In the experiments using COS‐7 cells expressing FLT3/ITD and Wt or mutant β‐catenin, FLT3/ITD phosphorylated Y654, and this residue was essential for β‐catenin’s nuclear localization by FLT3/ITD. Promoter‐reporter assays demonstrated that Y654 phosphorylation of β‐catenin was closely related to TCF transcriptional activity. In vitro kinase assays, using recombinant FLT3 and biotinylated β‐catenin peptide including Y654 showed that FLT3 directly phosphorylated Y654 of β‐catenin. These results explain how FLT3/ITD affects the tyrosine phosphorylation, nuclear localization, and transcriptional activity of β‐catenin. Targeting Y654 phosphorylation may lead to the development of novel approaches to therapy for FLT3/ITD‐AML.

Recurrent intramural hematoma of the small intestine in a severe hemophilia a patient with a high titer of factor VIII inhibitor: A case report and review of the literature

A 17-year-old man with severe hemophilia A (factor VIII <1%) developed intermittent left upper quadrant pain. He had a high titer of factor VIII inhibitor (1024 Bethesda units/mL) and was diagnosed with intramural hematoma of the jejunum. He was managed conservatively with activated prothrombin complex concentrate (APCC), resulting in the resolution of symptoms. He developed recurrent intramural hematoma of the small intestine over the next 54 months, and was successfully treated with APCC. This case highlights a rare clinical manifestation in hemophilia patients, and also indicates the effectiveness of APCC instead of exploratory surgery for intramural hematoma. Cases of intramural hematoma of the gastrointestinal tract among hemophilia patients are also reviewed.

Skewed X chromosome inactivation in fraternal female twins results in moderately severe and mild haemophilia B

Summary.  Female carriers of haemophilia B are usually asymptomatic; however, the disease resulting from different pathophysiological mechanisms has rarely been documented in females. In this study, we investigated the mechanisms responsible for haemophilia B in fraternal female twins. We sequenced the factor IX gene (F9) of the propositus, her father, a severe haemophilia B patient and the other family members. X chromosome inactivation was assessed by the methylation‐sensitive HpaII‐PCR assay using X‐linked polymorphisms in human phosphoglycerate kinase 1 gene (PGK1) and glutamate receptor ionotropic AMPA 3 gene (GRIA3). The twins were found to be heterozygotes with a nonsense mutation (p.Arg384X) inherited from their father. The propositus, more severely affected twin, exhibited a significantly higher percentage of inactivation in the maternally derived X chromosome carrying a normal F9. The other twin also showed a skewed maternal X inactivation, resulting in a patient with mild haemophilia B. Thus, the degree of skewing of maternal X inactivation is closely correlated with the coagulation parameters and the clinical phenotypes of the twins. Furthermore, we identified a crossing‐over in the Xq25–26 region of the maternal X chromosome of the more severely affected twin. This crossing‐over was absent in the other twin, consistent with their fraternal state. Differently skewed X inactivation in the fraternal female twins might cause moderately severe and mild haemophilia B phenotypes, respectively.

Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency.

INTRODUCTION Factor VII (FVII) is a vitamin K-dependent glycoprotein secreted into the blood circulation from hepatic cells. We investigated the molecular basis of the congenital FVII deficiency found in a Japanese patient. MATERIALS AND METHODS We analyzed the F7 gene of the patient, who was diagnosed with a FVII deficiency at pregnancy. We expressed a carboxyl-terminal truncated FVII (Arg462X FVII) corresponding to the identified mutation in CHO-K1 cells. To study roles of the carboxyl-terminus in the secretion of FVII, we also expressed a series of recombinant FVIIs deleted of limited numbers of carboxyl-terminal amino acids (462Arg-466Pro). RESULTS We identified a nonsense mutation (c.1384C>T: p.Arg462X) in F7, leading to a lack of five amino acids in the carboxyl-terminus. In expression experiments, Arg462X FVII was undetectable not only by Western blotting, but also by ELISA. A Western blot analysis of the truncated FVIIs revealed that all mutants were expressed in the cells the same as the wild type, but were secreted into the culture medium in lesser amounts than the wild type depending on the length of the deletion, which was confirmed by ELISA. Arg462X FVII did not colocalize with the Golgi on immunofluorescence staining, suggesting that it might be retained in the ER and degraded in the cell. CONCLUSION The carboxyl-terminal amino acids of FVII play an important role in its secretion, and the p.Arg462X mutation was likely to have caused the FVII deficiency in this patient.