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Naoki Itano

Publications and source records attributed to Naoki Itano.

16 recordsLinked to original sources
PubMed2006

SHAP potentiates the CD44-mediated leukocyte adhesion to the hyaluronan substratum.

CD44-hyaluronan (HA) interaction is involved in diverse physiological and pathological processes. Regulation of interacting avidity is well studied on CD44 but rarely on HA. We discovered a unique covalent modification of HA with a protein, SHAP, that corresponds to the heavy chains of inter-alpha-trypsin inhibitor family molecules circulating in blood. Formation of the SHAP.HA complex is often associated with inflammation, a well known process involving the CD44-HA interaction. We therefore examined the effect of SHAP on the CD44-HA interaction-mediated lymphocyte adhesion. Under both static and flowing conditions, Hut78 cells (CD44-positive) and CD44-transfected Jurkat cells (originally CD44-negative) adhered preferentially to the immobilized SHAP.HA complex than to HA. The enhanced adhesion is exclusively mediated by the CD44-HA interaction, because it was inhibited by HA, but not IalphaI, and was completely abolished by pretreating the cells with anti-CD44 antibodies. SHAP appears to potentiate the interaction by increasing the avidity of HA to CD44 and altering their distribution on cell surfaces. Large amounts of the SHAP.HA complex accumulate in the hyperplastic synovium of rheumatoid arthritis patients. Leukocytes infiltrated to the synovium were strongly positive for HA, SHAP, and CD44 on their surfaces, suggesting a role for the adhesion-enhancing effect of SHAP in pathogenesis.

Alpha-Globulins↗
PubMed2005

Hyaluronan synthase expression in pleural malignant mesotheliomas.

Hyaluronan (HA) is thought to play several important roles in tumor growth, tumorigenicity, and tumor dissemination and metastasis. Recently, three isoforms of hyaluronan synthase (HAS) have been cloned. Our objective was to determine which of the HAS isoforms were expressed in pleural malignant mesotheliomas, the most representative lesion of HA-producing tumors. We studied 10 cases of pleural malignant mesothelioma using novel antibodies of HAS. We compared HAS expression patterns of mesothelioma and pulmonary adenocarcinoma. Immunohistochemically, 9 of 10 (90%) cases of mesothelioma had extensive reaction to anti-HAS1 and anti-HAS2 antibodies, while HAS3 overexpression was present in 4 of 10 cases (40%). Of 20 cases of pulmonary adenocarcinoma, 5 overexpressed HAS1 (25%), 16 of 20 HAS2 (80%), and 4 of 20 HAS3 (20%). The expression level of HAS1 was significantly higher in mesotheliomas than in pulmonary adenocarcinoma (P=0.0036). Our data suggests that HAS1 might be a useful positive marker of malignant mesothelioma. However, a definitive conclusion should be based on further large-scale studies.

Adenocarcinoma↗
PubMed2005

Role of hyaluronan and hyaluronan synthase in endometrial cancer.

The aim of this study was to determine if the immunohistochemical expression of hyaluronan synthase (HAS) and serum levels of hyaluronan correlate with the clinicopathological manifestations of endometrial carcinoma. Sera were obtained from 59 endometrial cancer patients and 22 post-menopausal healthy women. Concentration of hyaluronan in sera was measured by an inhibitory ELISA using a hyaluronan-binding protein. Tissues obtained from 59 endometrial cancer patients were immunostained by the avidin-biotin-peroxidase complex method using anti-HAS1, anti-HAS2, anti-HAS3 and anti-CD44 antibody. A section was defined as having positive expression when >50% of the tumor cells were intensely stained. The expression of HAS1 was related to the depth of myometrial invasion, histological grade and lymph-vascular space involvement, but the expression of HAS2 and HAS3 was unrelated to these parameters. CD44 expression occurred more frequently in the HAS2- or HAS3-positive groups than in the HAS2- or HAS3-negative groups, and the expression of HAS1 was unrelated to CD44 expression. Serum levels of hyaluronan were higher in the endometrial cancer group than in the healthy control group, and increased with depth of myometrial invasion, histological grade and lymph-vascular space involvement. Serum hyaluronan levels were higher in the HAS1-positive group than in the HAS1-negative group, but the expression of HAS2 and HAS3 was unrelated to serum hyaluronan levels. HAS1 expression and an increase in serum hyaluronan in endometrial cancer may be associated with disease progression through myometrial invasion and lymph-vascular space involvement.

Adenocarcinoma↗
PubMed2004

Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction.

Hyaluronan is a major molecule in joint fluid and plays a crucial role in joint motion and the maintenance of joint homeostasis. The concentration and average molecular weight of hyaluronan in the joint fluids are reduced in osteoarthritis and rheumatoid arthritis. To elucidate the underlying mechanism, we analyzed the message expression of three isoforms of hyaluronan synthase and hyaluronidase from knee synovium, using real-time reverse transcriptase polymerase chain reaction. Synovia were obtained from 17 patients with osteoarthritis, 14 patients with rheumatoid arthritis, and 20 healthy control donors. The message expression of hyaluronan synthase-1 and -2 in the synovium of both types of arthritis was significantly less than in the control synovium, whereas that of hyaluronidase-2 in the synovium of both arthritides was significantly greater than in the control synovium. The decreased expression of the messages for hyaluronan synthase-1 and -2 and/or the increased expression of the message for hyaluronidase-2 may be reflected in the reduced concentration and decreased average molecular weight of hyaluronan in the joint fluids of patients with osteoarthritis and rheumatoid arthritis.

Adult↗
PubMed2004

A novel mechanism for the inhibition of hyaluronan biosynthesis by 4-methylumbelliferone.

Specific inhibitors of hyaluronan (HA) biosynthesis can be valuable therapeutic agents to prevent cancer invasion and metastasis. We have found previously that 4-methylumbelliferone (MU) inhibits HA synthesis in human skin fibroblasts and in group C Streptococcus. In this paper, the inhibition mechanism in mammalian cells was investigated using rat 3Y1 fibroblasts stably expressing HA synthase (HAS) 2. Exposure of the transfectants to the inhibitor resulted in significant reduction of HA biosynthesis and matrix formation. The evaluation of HAS transcripts and analysis of cell-free HA synthesis demonstrated the post-transcriptional suppression of HAS activity by MU. Most interesting, the post-transcriptional suppression of HAS activity was also observed using p-nitrophenol, a well known substrate for UDP-glucuronyltransferases (UGT). We investigated whether the inhibition was exerted by the glucuronidation of MU using both high pressure liquid chromatography and TLC analyses. The production of MU-glucuronic acid (GlcUA) was consistent with the inhibition of HA synthesis in HAS transfectants. MU-GlcUA was also detected at a similar level in control cells, suggesting that the glucuronidation was mediated by an endogenous UGT. Elevated levels of UGT significantly enhanced the inhibitory effects of MU. In contrast, the inhibition by MU was diminished to the control level when an excess of UDP-GlcUA was added to the cell-free HA synthesis system. We propose a novel mechanism for the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UGT resulting in a depletion of UDP-GlcUA.

Animals↗
PubMed2004

In vivo hyaluronan synthesis upon expression of the mammalian hyaluronan synthase gene in Drosophila.

Hyaluronan (HA) is a large linear polymer of repeating disaccharides of glucuronic acid and GlcNAc. Although HA is widely distributed in vertebrate animals, it has not been found in invertebrates, including insect species. Insects utilize chitin, a repeating beta-1,4-linked homopolymer of GlcNAc, as a major component of their exoskeleton. Recent studies illustrate the similarities in the biosynthetic mechanisms of HA and chitin and suggest that HA synthase (HAS) and chitin synthase have evolved from a common ancestral molecule. Although the biochemical properties and in vivo functions of HAS proteins have been extensively studied, the molecular basis for HA biosynthesis is not completely understood. For example, it is currently not clear if proper chain elongation and secretion of HA require other components in addition to HAS. Here, we demonstrate that a non-HA-synthesizing animal, the fruit fly Drosophila melanogaster, can produce HA in vivo when a single HAS protein is introduced. Expression of the mouse HAS2 gene in Drosophila tissues by the Gal4/UAS (upstream activating sequence) system resulted in massive HA accumulation in the extracellular space and caused various morphological defects. These morphological abnormalities were ascribed to disordered cell-cell communications due to accumulation of HA rather than disruption of heparan sulfate synthesis. We also show that adult wings with HA can hold a high level of water. These findings demonstrate that organisms synthesizing chitin (but not HA) are capable of producing HA that is structurally and functionally relevant to that in mammals. The ability of insect cells to produce HA supports the idea that in vivo HA biosynthesis does not require molecules other than the HAS protein. An alternative model is that Drosophila cells use endogenous components of the chitin biosynthetic machinery to produce and secrete HA.

Animals↗
PubMed2004

Selective expression and functional characteristics of three mammalian hyaluronan synthases in oncogenic malignant transformation.

Malignant transformation of fibroblasts and epithelial cells is often accompanied by increased hyaluronan production and accumulation. Despite recent progress in the study of hyaluronan biosynthesis, the mechanisms underlying the transformation-induced overproduction of hyaluronan have not been elucidated. Here we report that activity and transcriptional levels of hyaluronan synthase (HAS) significantly increased after oncogenic malignant transformation of a rat 3Y1 fibroblast cell line. Of three HAS isoforms (HAS1, HAS2, and HAS3), only HAS2 gene expression was increased in the v-Ha-ras transformed 3Y1 cells, which show less malignancy. In contrast, both HAS1 and HAS2 expressions were elevated in the highly malignant cells transformed with v-src and/or v-fos. To assess the contribution of HAS expression to the oncogenic malignant transformation, we established stable cell transfectants expressing sense and antisense HAS genes. Antisense suppression of the HAS2 expression significantly decreased hyaluronan production in the cells transformed by the oncogenic v-Ha-ras and eventually led to a reduction in tumorigenicity in the rat peritoneum. The introduction of the HAS1 and HAS2 genes promoted the growth of subcutaneous tumors in a manner dependent on the levels of hyaluronan synthesis. Significant growth promotion was observed within a wide range of HAS1 expression. In contrast, the growth stimulation was only seen within a narrow range of HAS2 expression, and high levels of HAS2 expression even inhibited tumor growth. These results suggest that proper regulation of the expression of each HAS isoform is required for optimal malignant transformation and tumor growth.

Animals↗
PubMed2004

Elevated transcript level of hyaluronan synthase1 gene correlates with poor prognosis of human colon cancer.

Hyaluronan plays important roles in the complex processes of tumor invasion and metastasis. It is now known that three hyaluronan synthase (HAS) isoforms catalyze hyaluronan synthesis, which raises the question of how they are involved in malignant tumor progression. In this study, we examined the correlation between tumor progression and transcriptional levels of three HAS isoforms in specimens of human colon cancers. Tumor tissues from 31 patients with different diagnostic grades were assessed to determine the level of each HAS isoform by real time RT-PCR. The mean expression coefficients for HAS1, HAS2 and HAS3 in the cancerous parts were 0.82-, 0.91- and 1.22-fold, respectively; of those in the noncancerous parts at Dukes' stage A; 1.00-, 0.95- and 1.06-fold, respectively, at stage B; and 1.95-, 1.16- and 1.19-fold, respectively, at stage C. In survival analysis, a significant correlation was observed between poor survival and the HAS1 transcript level. When the ratio of tumor to normal tissue in the HAS1 level was compared with that of the HA receptor transcript level, there was a positive correlation with that of the CD44 variant 6 level at Dukes' stage C. Our current results therefore suggest that HAS1 plays a role in the malignant progression of human colon cancer cells.

Base Sequence↗
PubMed2004

Differential regulation by IL-1beta and EGF of expression of three different hyaluronan synthases in oral mucosal epithelial cells and fibroblasts and dermal fibroblasts: quantitative analysis using real-time RT-PCR.

Using "real-time RT-PCR", we assessed the expression of three different hyaluronan synthase genes, HAS1, HAS2, and HAS3, by measuring their mRNA amounts in cultured human oral mucosal epithelial (COME) cells, oral mucosal fibroblasts, and dermal fibroblasts, and investigated the effects of interleukin-1beta (IL-1beta) and epidermal growth factor (EGF). When COME cells were treated with IL-1beta or EGF, early and marked increases and subsequent rapid decreases were observed for all HAS genes and, moreover, actual changes in hyaluronan synthesis subsequently occurred. The effects of IL-1beta stimulation were concentration-dependent and the maximal response to the EGF stimulation was observed at a low concentration (0.1 ng per mL). When two different types of fibroblasts were treated with IL-1beta or EGF, increased expression with different degrees and rates of three different HAS genes and subsequent increased synthesis of hyaluronan were also observed. In addition, HAS1 gene expression was not detectable in the mucosal fibroblasts, while weak HAS3 gene expression was detected in the dermal fibroblasts. Taken together, it is likely that the regulation of the expression of the three different HAS genes is different between oral mucosa and skin, which may be of significance for elucidating some of the differences between these tissues in wound healing.

Cells, Cultured↗
PubMed2004

Hyaluronan synthase expression in ovarian cancer.

To determine if the immunohistochemical expression of hyaluronan synthase (HAS) correlates with the clinicopathological manifestations or clinical outcomes of ovarian carcinoma, sections of tumor tissue from 33 ovarian cancer patients were immunostained by the avidin-biotin-peroxidase complex method using anti-HAS1, anti-HAS2, anti-HAS3 and anti-CD44 antibody. A section was defined as having positive expression when >50% of the tumor cells were intensely stained. The microvessel density, which was defined as the mean number of new vessels, was determined under light microscopy. In the 33 ovarian cancer cases, 12 cases had positive expression of HAS1, 21 cases had positive expression of HAS2 and 11 cases had positive expression of HAS3. The expression of HAS1, HAS2 and HAS3 was unrelated to the stage of disease. CD44 expression occurred more frequently in the HAS1-positive group than in the HAS1-negative group, but the expression of HAS2 and HAS3 was unrelated to CD44 expression. The microvessel density was higher in the HAS1-positive group than in the HAS1-negative group. But the microvessel density did not differ in relation to the expression of HAS2 and HAS3. In the 23 patients that received chemotherapy, the expression of HAS1, HAS2 and HAS3 was unrelated to the chemotherapy response. The overall survival time was longer in the HAS1-negative group than in the HAS1-positive group. However, the expression of HAS2 and HAS3 was unrelated to the overall survival time. These results suggest that HAS1 expression in ovarian cancer may be associated with disease progression through angiogenesis and is an independent predictor of patient survival.

Cystadenocarcinoma, Serous↗
PubMed2003

Cell spreading controls endoplasmic and nuclear calcium: a physical gene regulation pathway from the cell surface to the nucleus.

Cells attaching to an extracellular matrix through integrins flatten out (spread) on the matrix, eliciting cellular signals needed for survival. We show that the shape of the nucleus changes and the nuclear calcium level increases in spreading cells. Moreover, cell spreading and osmotic stretching of isolated nuclei cause release of perinuclear Ca(2+), and patch clamping of nuclei reveals stretch-activated Ca(2+) permeable channels. Gene expression assays with myocyte enhancer factor 2, which is activated by calmodulin-dependent kinase IV, indicate that the elevation in nuclear Ca(2+) is functionally significant. We propose a mechano-transduction pathway in which spreading-induced nuclear stretching releases Ca(2+) from the perinuclear space, and the resulting Ca(2+) elevation in the nucleus provokes changes in gene expression.

3T3 Cells↗
PubMed2002

The role of syndecan-2 in regulation of actin-cytoskeletal organization of Lewis lung carcinoma-derived metastatic clones.

Syndecans, a family of transmembrane heparan sulphate proteoglycans, contribute to various biological processes, including adhesion, motility, proliferation, differentiation and morphogenesis. We document here the involvement of syndecan-2 acting alone or co-operatively with integrin alpha5beta1, for regulation of actin-cytoskeletal organization on cell adhesion to fibronectin, using fibronectin-recombinant polypeptides containing the ligands for either or both of these receptors as substrata. Lewis lung carcinoma-derived low-metastatic P29 cells binding to the substrata by both receptors formed actin stress fibres, whereas those binding by syndecan-2 or integrin alpha5beta1 alone formed filopodia or cortex actin. In contrast, higher metastatic LM66-H11 cells formed cortex actin even on substrata containing both ligands. Northern-blot and flow-cytometric analyses revealed that syndecan-2 expression in LM66-H11 cells was significantly lower (1/4.5 in mRNA and 1/8 in cell-surface expression) than in P29 cells, whereas expression levels of integrin alpha5beta1 and other syndecans were similar in both cell types. These results suggest that the failure of LM66-H11 to form stress fibres is due to a lower expression of syndecan-2 than that due to a threshold for its function. This was confirmed by the finding that overexpression of syndecan-2 by transfection of its cDNA into LM66-H11 cells caused the formation of stress fibres on the fibronectin substratum. These in vitro cellular responses of the two clones might reflect their in vivo situation in primary tumours in which P29 cells with a stroma-inducing capacity were immediately surrounded by fibronectin-rich matrix formed by the induced stromal cells, whereas LM66-H11 cells without such capacity were not surrounded by a similar matrix.

Actins↗
PubMed2002

Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration.

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.

Agar↗
PubMed2002

Mammalian hyaluronan synthases.

Three mammalian hyaluronan (HA) synthase genes, HAS1, HAS2, and HAS3, have been cloned and expressed, allowing the mechanisms for regulation of HA biosynthesis and function to be studied. The hyaluronan synthase (HAS) isoforms differ in kinetic characteristics and product size. The expression of each HAS isoform is controlled in a different fashion when mammalian cells are stimulated by various cytokines and the expression patterns are both spatially and temporally regulated during embryonic development. The existence of three different HAS isoforms with different characteristics implies that the broad range of biological and physiological roles performed by HA are regulated by controlling the activities and expression of the HAS isoforms. This review focuses on recent findings on the regulatory mechanisms for controlling HA biosynthesis and provides new insights into the enzymic basis for the functional regulation of HA.

Animals↗
PubMedDate not supplied

CD44 variant exon 6 expressions in colon cancer assessed by quantitative analysis using real time reverse transcriptase-polymerase chain reaction.

CD44 is a family of transmembrane glycoproteins that serve as a major receptor for hyaluronate and the splice variants play a very important role in tumor progression and metastasis. We examined the relationship between cancer progression and mRNA levels of CD44 variant exon 6 (CD44v6) in specimens of colon cancer at different diagnostic stages from 31 patients using real time RT-PCR analysis. Increased mRNA levels of CD44v6 were observed in 82% of the specimens in comparison with those in the corresponding non-cancerous tissue specimens. A statistically significant correlation between the CD44v6 expression and the cancerous state was found in most specimens at all Dukes stages. None of the other parameters were related to the expression in the cancerous specimens. Quantitative real time RT-PCR analysis showed that there was no correlation of CD44v6 expression with tumor progression, although CD44v6 is upregulated in transformation. Thus, CD44v6 expression may be a clinically useful indicator of colon cancer.

Aged↗