Minoru Matsui

Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression.

Recent works have shown the importance of reduction/oxidation (redox) regulation in various biological phenomena. Thioredoxin (TRX) is one of the major components of the thiol reducing system and plays multiple roles in cellular processes such as proliferation, apoptosis, and gene expression. To investigate the molecular mechanism of TRX action, we used a yeast two-hybrid system to identify TRX-binding proteins. One of the candidates, designated as thioredoxin-binding protein-2 (TBP-2), was identical to vitamin D3 up-regulated protein 1 (VDUP1). The association of TRX with TBP-2/VDUP1 was observed in vitro and in vivo. TBP-2/VDUP1 bound to reduced TRX but not to oxidized TRX nor to mutant TRX, in which two redox active cysteine residues are substituted by serine. Thus, the catalytic center of TRX seems to be important for the interaction. Insulin reducing activity of TRX was inhibited by the addition of recombinant TBP-2/VDUP1 protein in vitro. In COS-7 and HEK293 cells transiently transfected with TBP-2/VDUP1 expression vector, decrease of insulin reducing activity of TRX and diminishment of TRX expression was observed. These results suggested that TBP-2/VDUP1 serves as a negative regulator of the biological function and expression of TRX. Treatment of HL-60 cells with 1α,25-dihydroxyvitamin D3 caused an increase of TBP-2/VDUP1 expression and down-regulation of the expression and the reducing activity of TRX. Therefore, the TRX-TBP-2/VDUP1 interaction may be an important redox regulatory mechanism in cellular processes, including differentiation of myeloid and macrophage lineages.

Expression of thioredoxin in bleomycin-injured airway epithelium: possible role of protection against bleomycin induced epithelial injury.

Bleomycin (BLM) is an anticancer drug, administration of which leads to severe lung injury, in which the generation of intracellular reactive oxygen species (ROS) is thought to participate in that. Thioredoxin (TRX) has been found to function as a powerful antioxidant by reducing ROS, and thus protecting against ROS-mediated cytotoxicity. However, a protective role of TRX in BLM-induced lung injury has not been determined. In the present study, we therefore attempted to clarify this issue. Human TRX-transfected L929 murine fibrosarcoma cells were more resistant to BLM-induced cytotoxicity than the parental and the control transfected cells, indicating that TRX plays the protective role in BLM-induced cytotoxicity. Next, we examined TRX expression in the lung of in vivo model of BLM-induced lung injury and BLM-stimulated bronchial epithelial cells in vitro to clarify the role of TRX in BLM-induced lung injury. In the lungs of BLM-treated mice, the expression of TRX was strongly induced in bronchial epithelial cells. TRX expression was also up-regulated at both the mRNA and protein levels in cultured BEC with the treatment with BLM. However, the expression of other major antioxidants, such as Cu/Zn-SOD, Mn-SOD, catalase and glutathione peroxidase, was not affected by BLM. These observations suggest that the cellular reduction and oxidation (redox) state modified by TRX is involved in the BLM resistancy and the induction of TRX in bronchial epithelial cells might play a protective role in BLM-induced lung injury.

Identification of novel CD23 transcripts on human T and B lymphocytes and eosinophil cell line.

The main aim of the present studies was to investigate the structure of the human low-affinity IgE Fc receptor (CD23) present on T and B lymphoid cells and eosinophil cell line. A novel finding in these studies has been the detection and sequence analysis of CD23 transcripts in human T lymphocytes. These studies have established that some of the human T-cell populations analyzed express CD23 mRNA and that its structure is quite similar to that previously described for human B lymphocytes. A second major finding in these studies is that some human T- and B-cell lines and eosinophil cell line contain multiple forms of CD23 transcripts. These appear to be generated via alternative splicing, resulting in transcripts that may encode a truncated, possibly secretory form of CD23. These findings in human T and B lymphocytes and eosinophils provide new information about the structure of lymphocyte CD23 and suggest that alternative processing of transcripts generates CD23 mRNA that encodes CD23 isoforms. These studies are the first experimental evidence showing that CD23 isoforms may occur in the human and are the first direct evidence for production of CD23 by human T lymphocytes. In addition, these studies provide the first experimental evidence that T and B lymphocytes express CD23 transcripts lacking exon 3-encoded sequences, raising the possibility that a secretory form of CD23 may be synthesized by human T and B lymphocytes, and eosinophils.

Structure of the mouse thioredoxin-encoding gene and its processed pseudogene

Thioredoxins (TXN) are small proteins with various biological functions, such as redox regulation, found in many species including bacteria, plants and animals. We previously reported the isolation of the TXN-encoding cDNAs from human and mouse. In order to elucidate the functions of the mammalian TXN system, we planned to generate Txn knockout mice, and cloned the genomic DNA fragments using the Txn cDNA as a probe. The Txn gene extends over 12 kb and consists of five exons separated by four introns. Detailed Southern analyses revealed that the mouse genome contains only one active Txn gene and one processed pseudogene (Txn-ps1), in contrast to some species which have families of active TXN-encoding genes. These findings should help to understand Txn itself, and provide a basis for transgenic experiments by gene targeting.

Alternative transcripts of the human CD23/FcϵRII: A possible novel mechanism of generating a soluble isoform in the type-II cell surface receptor

Human CD23/Fc epsilon RII is a 45 kDa type-II membrane glycoprotein having two isoforms (a and b) that only differ in the structures of their intracytoplasmic tails. CD23/Fc epsilon RII has been demonstrated to have multiple roles in the immune system such as regulation of lymphocyte growth and differentiation and IgE-mediated immune responses. Here, we found that the human B-cell line RPMI8866, in addition to a and b transcripts, contained shorter transcripts (a and b) that lack the entire third exon. These alternative transcripts were also detected in peripheral blood lymphocytes as well as other hematopoietic cell lines with CD23/Fc epsilon RII. Because exon 3 encodes all of the transmembrane segment and the anchoring region of the cytoplasmic tail, it is suggested that a and b transcripts encode secretory forms of CD23/Fc epsilon RII or they may function as regulatory transcripts involved in the control of CD23/Fc epsilon RII expression.Human CD23/FcϵRII is a 45 kDa type‐II membrane glycoprotein having two isoforms (a and b) that only differ in the structures of their intracytoplasmic tails. CD23/FcϵRII has been demonstrated to have multiple roles in the immune system such as regulation of lymphocyte growth and differentiation and IgE‐mediated immune responses. Here, we found that the human B‐cell line RPMI8866, in addition to a and b transcripts, contained shorter transcripts (a and b) that lack the entire third exon. These alternative transcripts were also detected in peripheral blood lymphocytes as well as other hematopoietic cell lines with CD23/FcϵRII. Because exon 3 encodes all of the transmembrane segment and the anchoring region of the cytoplasmic tail, it is suggested that a and b transcripts encode secretory forms of CD23/FcϵRII or they may function as regulatory transcripts involved in the control of CD23/FcϵRII expression.

IL-10 suppresses cell surface CD23/FcεRII expression, not by enhancing soluble CD23 release, but by reducing CD23 mRNA expression in human monocytes

To examine a possible involvement of interleukin-10 (IL-10) in CD23/FcεRII expression in human monocytes, effects of IL-10 on the cell surface CD23 expression, soluble CD23 (sCD23) release, and CD23 type b mRNA expression were investigated. IL-10 suppressed IL-4-induced surface CD23 expression on monocytes in a dose-dependent manner, and this effect was completely neutralized by anti-IL-10 antibody. The suppressive effect of IL-10 on surface CD23 expression was not due to enhancement of sCD23 release from the cell surface because no increase in sCD23 in culture supernatant was detected after incubation with IL-10. Instead, the effect of IL-10 seemed to be exerted at the transcriptional level since IL-4-induced expression of CD23 type b mRNA was significantly reduced when IL-10 was present. Although IL-4 induced surface CD23 expression on both monocytes and B cells, the suppressive effect of IL-10 was observed only on monocytes, which underscores different regulatory mechanisms for CD23 expression between the two cell types.

Characterization of novel FcϵRII/CD23 isoforms lacking the transmembrane (TM) segment in human cell lines

Human FcepsilonRII/CD23 is an approximately 45 kDa type II transmembrane glycoprotein belonging to the C-type animal-lectin family, and has two isoforms (a and b) that only differ in their intracytoplasmic tails. We previously found that in several human and mouse cell lines there were two additional CD23 transcripts (a and b) lacking the exon 3 that encodes the entire transmembrane segment and a part of cytoplasmic tails. In this study, we analyzed the putative CD23a and CD23b products at protein levels and characterized with rabbit polyclonal antibodies against novel amino-acid sequences of the putative CD23a and CD23b molecules (anti-CD23a Ab, anti-CD23b Ab). Western blots in COS cells transfected with CD23a or CD23b cDNA as well as in vitro translation assays showed that the a and b CD23 transcripts were translated to about 40 kDa molecules. These 40 kDa molecules were also recognized by a polyclonal antibody against 25 kDa soluble fragment of human CD23. We also found that human cells having mRNAs for CD23a and CD23b expressed protein products recognized specifically by anti-CD23a or anti-CD23b Ab, respectively. In addition, the CD23a and CD23b molecules in transfected COS cells were resistant to Endo H(f) and PNGase F, although these truncated forms as well as the membrane-associated forms had an asparagine residue responsible for the N-linked glycosylation. Taken together, our results show that the a and b CD23 transcripts are expressed and translated in human lymphoid cells and that their translated products are retained in the cytoplasm where they might play an unique regulatory role in the expression of the full-length CD23 on the cell surface.