Ken-ichi Matsumoto

Extracellular matrix tenascin-X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation.

An extracellular matrix tenascin‐X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels. The precise in vivo role of TNX remains to be elucidated. To identify proteins that interact with TNX in the extracellular environment, we searched for TNX‐binding proteins using a yeast two‐hybrid system.

Tumour invasion and metastasis are promoted in mice deficient in tenascin-X.

Background Tenascin‐X (TNX) is a member of the tenascin family of large oligomeric glycoproteins of the extracellular matrix (ECM). To determine whether TNX plays a part in tumour invasion and metastasis and to disclose its normal physiological role, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in TNX.

Structural analysis of mouse tenascin-X: evolutionary aspects of reduplication of FNIII repeats in the tenascin gene family

Tenascin-X (TNX) is an extracellular matrix glycoprotein involved in both primary structural functions and modulating cellular activities in multicellular organisms. We determined the 67977bp nucleotide sequence of the entire mouse tenascin-X (Tnx) gene, which also includes the last exon of Creb-rp and Cyp21. We compared it with the orthologous human locus. Conservation of both position and orientation of the three functionally unrelated genes at this position was found. Comparison also revealed that introns 1, 4 and 6 of Tnx are highly conserved between species. The sequence showed that mouse Tnx contains 43 exons separated by 42 introns. The deduced amino-acid sequence (4114 residues) revealed that mouse Tnx has a primary structure characteristic of tenascins, which consists of a signal peptide and four heptad repeats followed by 18.5 epidermal growth factor-like (EGF) repeats, 31 fibronectin type III-like (FNIII) repeats, and a region homologous to fibrinogen. cDNA clones generated by alternative splicing of eight consecutive FNIII repeats (M15-M22) as well as a proximal FNIII repeat (M3) were also identified. The FNIII motifs that were subject to alternative splicing were assigned to the group of recently reduplicated FNIII repeats because they have a high level of amino-acid sequence similarity. We also analyzed the evolution of FNIII repeats in TNX.

DJ-1, a causative gene product of a familial form of Parkinson's disease, is secreted through microdomains

DJ‐1 is secreted into the serum and plasma of patients with various diseases. In this study, DJ‐1 was found to be secreted into culture media of various cells and the amount of wild‐type DJ‐1 secreted was two‐fold greater than that of mutant DJ‐1 of cysteine at 106 (C106). Furthermore, the oxidative status of more than 90% of the DJ‐1 secreted from HeLa cells was SOH and SO2H forms of C106. A portion of DJ‐1 in cells was localized in microdomains of the membrane. These findings suggest that DJ‐1 is secreted through microdomains and that oxidation of DJ‐1 at C106 facilitates the secretion.

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1.

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.

Induction of truncated form of tenascin-X (XB-S) through dissociation of HDAC1 from SP-1/HDAC1 complex in response to hypoxic conditions

XB-S is an amino-terminal truncated protein of tenascin-X (TNX) in humans. The levels of the XB-S transcript, but not those of TNX transcripts, were increased upon hypoxia. We identified a critical hypoxia-responsive element (HRE) localized to a GT-rich element positioned from -1410 to -1368 in the XB-S promoter. Using an electrophoretic mobility shift assay (EMSA), we found that the HRE forms a DNA-protein complex with Sp1 and that GG positioned in -1379 and -1378 is essential for the binding of the nuclear complex. Transfection experiments in SL2 cells, an Sp1-deficient model system, with an Sp1 expression vector demonstrated that the region from -1380 to -1371, an HRE, is sufficient for efficient activation of the XB-S promoter upon hypoxia. The EMSA and a chromatin immunoprecipitation (ChIP) assay showed that Sp1 together with the transcriptional repressor histone deacetylase 1 (HDAC1) binds to the HRE of the XB-S promoter under normoxia and that hypoxia causes dissociation of HDAC1 from the Sp1/HDAC1 complex. The HRE promoter activity was induced in the presence of a histone deacetylase inhibitor, trichostatin A, even under normoxia. Our results indicate that the hypoxia-induced activation of the XB-S promoter is regulated through dissociation of HDAC1 from an Sp1-binding HRE site.

Invasion of Melanoma in Double Knockout Mice Lacking Tenascin‐X and Tenascin‐C

The roles of extracellular matrix glycoproteins belonging to the tenascin family in the regulation of tumor cell proliferation, invasion, and metastasis are not known. To address this issue, we generated tenascin‐X (TNX) and tenascin‐C (TNC) double knockout mice and compared findings in these mice with those in single knockout (TNX+/+TNC‐/‐ and TNX‐/‐TNC+/+) mice. We investigated the proliferation and invasion of B16‐BL6 melanoma cells after these cells had been injected into the footpads of mice in each group. The primary tumor size and invasion to the ankle adjacent to the primary tumor site were examined at 35 days after injection of the melanoma cells. The primary tumor size in TNX‐/‐TNC+/+ mice was significantly larger than that in wild‐type mice, but those of TNX+/+TNC‐/‐ and double knockout mice were comparable to that in the wild‐type mice. On the other hand, invasion to the ankle was obviously promoted in TNX‐I‐ TNC+/+ and double knockout mice compared with that in the wild‐type mice, but invasion to the ankle in TNX+/+TNC‐/‐ mice was only slightly promoted. Gelatin zymography confirmed increased matrix metalloproteinase (MMP)‐9 activity in the dorsal skin of TNX‐/‐TNC+/+, TNX+/+TNC‐/‐ and double knockout mice. However, the amounts of MMP‐9 mRNA in the dorsal skins of all mice were almost the same, indicating that the increased activity of MMP‐9 in the single and double knockout mice is regulated at the MMP‐9 processing level. These results indicate that MMP‐9 is activated in all TN‐deficient mice, but that TNX exerts a greater effect on tumor invasion than does TNC.

Disruption of MSSP, c‐myc single‐strand binding protein, leads to embryonic lethality in some homozygous mice

Background MSSP, c‐myc single‐strand binding protein, works as a factor for DNA replication, transcription, apoptosis induction, and myc/ras cooperative transformation. The cDNAs encoding four of the family proteins, MSSP‐1, MSSP‐2, Scr2 and Scr3, were cloned. These proteins possess two copies of putative RNA binding domains, RNP‐A and RNP‐B, and these RNA binding domains have been suggested to be indispensable to the functions of MSSP.

Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice.

Tenascin‐X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX‐deficient mice in comparison with that of wild‐type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX‐deficient (TNX−/−) mice. By thin‐layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX−/− mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX−/− mice. By gas chromatography‐mass spectrometry analysis of triglyceride‐associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX−/− mice compared with those in wild‐type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock‐transfected cells were also caused by over‐expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride‐associated fatty acids.

Truncated form of tenascin-X, XB-S, interacts with mitotic motor kinesin Eg5

XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5.