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T Hasunuma

Publications and source records attributed to T Hasunuma.

At least 19 recordsLinked to original sources
PubMed2001

Potential role of HOXD9 in synoviocyte proliferation.

OBJECTIVE: To investigate the role of HOXD9 in the proliferation activity of cultured synoviocytes as well as the mechanisms that regulate HOXD9 transcription. METHODS: Synoviocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were transfected with HOXD9 complementary DNA to establish stable transformants that overexpressed HOXD9. HOXD9 expression was detected by Western blotting with anti-HOXD9 antibody. The growth properties of the transformants were investigated by proliferation and colony formation assays. The expression of basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNFalpha), interleukin-1beta, c-Fos, and c-Myc was examined by Western blotting. Transcriptional regulation of HOXD9 was examined by transient cotransfection. RESULTS: HOXD9 protein was highly expressed in RA synoviocytes, but there was no expression in OA synoviocytes. HOXD9 transfection induced stable HOXD9 protein expression in synoviocytes and showed an increased proliferation rate under both normal and serum-starved conditions, as well as an enhanced capacity to proliferate anchorage independently to form colonies in soft agar cultures, compared with control transfectants. Higher levels of bFGF and c-Fos were detected in HOXD9 transformants than in controls. Transient cotransfection assays of NIH3T3 fibroblasts and synoviocytes showed that HOXD9 activated the luciferase reporter construct containing the highly conserved region (HCR), an autoregulatory element of HOXD9 promoter. This activation was significantly increased by bFGF, suppressed by TNFalpha, and unchanged by transforming growth factor beta in synoviocytes. Human T lymphotropic virus type I tax also activated the luciferase reporter construct containing the HCR and had a synergistic effect with HOXD9 on HCR promoter activation. CONCLUSION: Our data suggest that HOXD9 plays a potential role in synovial proliferation. In addition, they suggest that the involvement of HOXD9 in the regulation of cellular growth might be mediated, at least in part, by up-regulation of growth-related factors such as bFGF and c-Fos and/or might result from increased transcription activity by its regulators.

3T3 Cells↗
PubMed2001

Infection of synoviocytes with HTLV-I induces telomerase activity.

To investigate the mechanism of synovial hyperplasia by human T-lymphotropic virus type I (HTLV-I) infection, the enzymatic activity of telomerase and expression of telomerase-related factors in HTLV-I infected synoviocytes were examined. Cultured synoviocytes obtained from four patients with osteoarthritis (OA) and four with traumatic joint disease (TJD) were infected by HTLV-I. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of telomerase-related mRNAs such as telomerase reverse transcriptase (hTERT), telomerase RNA component (hTERC), and telomeric repeat binding factor 2 (TRF2) were also examined. Telomerase activity was detected in all HTLV-I-infected synoviocytes but not in uninfected synoviocytes. A remarkable induction of hTERT mRNA was observed in four of eight HTLV-I-infected synoviocytes, whereas expressions of hTERC, TRF2, and TEP-1 mRNAs were not changed. Our results clearly demonstrate that HTLV-I upregulates telomerase activity in synoviocytes probably via upregulation of hTERT activity. These findings suggest that telomerase activation in synoviocytes has an important role in upregulated proliferative activity of HAAP synoviocytes.

Carrier Proteins↗
PubMed2000

CBP: A target molecule of HTLV-1 Tax in synoviocyte activation.

Previous studies have shown that human T-cell leukemia virus type 1 (HTLV-1) Tax is a key molecule of synoviocyte activation in HTLV-1 associated arthropathy (HAAP). To clarify the molecular mechanism of HTLV-1 Tax-induced transcriptional activation in synoviocytes from HAAP, we investigated the role of cyclicAMP (cAMP)-regulated enhancer (CRE) binding protein (CREB)-binding protein (CBP), as a target molecule of HTLV-1 Tax. Activation of cyclic AMP (cAMP)/protein kinase-A (PK-A) pathway resulted in a significantly high response of CRE promoter in synoviocytes from patients with HAAP as well as in Tax-transiently transfected synoviocytes from patients with rheumatoid arthritis (RA). Mammalian two-hybrid analysis showed that the recruitment of CBP was responsible for CREB activation. Furthermore, PK-A activation induced CBP-Tax complex in synoviocytes from HAAP and the complex contained CREB. These findings demonstrated that complex formation of CBP and Tax is critical for enhanced CREB activity in synoviocytes from HAAP.

CREB-Binding Protein↗
PubMed2000

Caspase-9 regulates cisplatin-induced apoptosis in human head and neck squamous cell carcinoma cells.

The aim of this study was to understand the molecular requirements for cisplatin-induced apoptosis in human head and neck squamous cell carcinoma (HNSCC) cell death. Cisplatin induced apoptosis in HNSCC cell lines, HSC-2, HSC-3 and HSC-4 in a dose-dependent manner. However, cisplatin did not induce the expression of Fas-ligand mRNA or upregulation of Fas protein. By caspase activation assays, cisplatin induced Caspase-3 (Casp-3), -8 and -9 activation. In all three lines tested, the use of a specific inhibitor of Casp-9 almost completely blocked cisplatin-induced apoptosis, while the use of Casp-3 and -8 inhibitors resulted in a partial blockade of cisplatin-induced apoptosis. Our results strongly suggest that Casp-9-dependent apoptosis plays an important role in cisplatin-induced HNSCC apoptosis.

Apoptosis↗
PubMed2000

Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules.

OBJECTIVE: Fas-mediated apoptosis is associated with the pathophysiology of rheumatoid arthritis (RA). However, the molecular mechanisms of this process remain to be elucidated in rheumatoid synovium. We investigated the behavior of intracellular signaling molecules that regulate Fas-mediated apoptosis in RA synoviocytes. METHODS: Anti-Fas monoclonal antibody (mAb) was added to RA synoviocytes after treatment with tumor necrosis factor alpha (TNFalpha) or basic fibroblast growth factor (bFGF) for 5 days. The cytotoxic activity was measured using a lactate dehydrogenase-release assay. The expression of apoptosis-related molecules in RA synoviocytes was examined by immunoblot analysis. The enzymatic activities of caspases 3 and 8 under Fas ligation were examined. Transfer of the FADD (Fas-associated death domain) protein and the FLIP(L) (long form of the FLICE [FADD-like interleukin-1beta-converting enzyme]-inhibitory protein) gene into RA synoviocytes was performed using adenoviral vectors. RESULTS: Following a 5-day treatment with TNFalpha or bFGF, Fas ligation with its agonistic mAb induced apoptosis of almost all TNFalpha-treated RA synoviocytes but only showed a weak apoptotic activity in bFGF-treated synoviocytes. Although there was no correlation between the induction of Fas-mediated apoptosis and the expression of apoptosis-related molecules among these cells, a high enzymatic activity of caspases 3 and 8 was observed only in the TNFalpha-treated RA synoviocytes after Fas ligation. The bFGF-treated RA synoviocytes were relatively resistant to apoptosis induced by FADD gene transfection, as compared with the TNFalpha-treated synoviocytes. In addition, the expression of FLIP(L), an inhibitory molecule of Fas-mediated apoptosis, was reduced in TNFalpha-treated RA synoviocytes, and the expression of FLIP43 was augmented in bFGF-treated RA synoviocytes. Moreover, Fas-mediated apoptosis in TNFalpha-treated RA synoviocytes was partially inhibited by FLIP(L) gene transfection. CONCLUSION: Our results suggest that Fas-mediated apoptosis of RA synoviocytes is differentially regulated by TNFalpha and bFGF. In addition, the regulatory mechanisms of apoptosis involve the formation of the death-inducing signaling complex, especially at the level of caspase 8 activation, and this process may be partly associated with FLIP expression.

Antibodies, Monoclonal↗
PubMed2000

Basic FGF-induced activation of telomerase in rheumatoid synoviocytes.

To investigate the mechanism of persistent proliferation of rheumatoid arthritis (RA) synoviocytes in situ, we examined the activity of telomerase enzyme and the expression of telomerase related factors in cultured synoviocytes. Cultured synoviocytes obtained from patients with rheumatoid arthritis (n = 29), osteoarthritis (OA, n = 18), and traumatic joint disease (TJD, n = 4) were examined. Telomerase activity was detected by TRAP (telomeric repeat amplification protocol) assay, and 12 out of 29 samples of synoviocytes (41%) from RA patients showed a positive telomerase activity, whereas none of the samples from OA and TJD patients showed this activity. Results were confirmed by PCR-ELISA. The telomerase activity was enhanced by basic fibroblast growth factor (bFGF). The mRNA expression of telomerase related factors, such as hTERC, TRF2, and TEP-1, showed no difference between RA and OA synoviocytes. Our results suggest that telomerase is activated in rheumatoid synoviocytes, and that bFGF upregulates the activity of this enzyme in RA synoviocytes.

Arthritis, Rheumatoid↗
PubMed2000

Novel gene therapy for rheumatoid arthritis by FADD gene transfer: induction of apoptosis of rheumatoid synoviocytes but not chondrocytes.

Synovial cells in the rheumatoid synovium show abnormal proliferation, leading to joint destruction. Rheumatoid synovial cells express functional Fas antigen and are susceptible to Fas-mediated apoptosis. We have proposed the induction of apoptosis by Fas/Fas ligand system of proliferative rheumatoid synovium as a novel therapy for rheumatoid arthritis (RA). We have recently reported that Fas-associated death domain protein (FADD) plays a key role in Fas-mediated apoptosis of synovial cells in patients with RA. In this study, we determined whether FADD gene transfer could induce apoptosis of RA synoviocytes in vitro and in vivo. Transfection of FADD gene by adenoviral vector into cultured RA synoviocytes induced up-regulation of FADD expression and apoptosis. In addition, local injection of FADD adenovirus (Ad-FADD) eliminated synoviocytes in vivo by induction of apoptosis of proliferating human rheumatoid synovium engrafted in severe combined immunodeficiency mouse, which is the most suitable animal model of RA for the evaluation of treatment strategy in vivo. In addition, Ad-FADD-induced apoptosis was limited to cells of the synovium tissue and did not affect chondrocytes. Our results strongly suggest that FADD gene transfer can induce apoptosis of RA synoviocytes both in vitro and in vivo, suggesting that FADD gene transfer might be effective in the treatment of RA.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Fas-associated death domain protein is a Fas-mediated apoptosis modulator in synoviocytes.

OBJECTIVE: To understand the intracellular regulatory mechanisms in Fas-mediated apoptosis of synoviocytes, we examined the involvement of caspases [caspase-1/ICE (interleukin-1beta converting enzyme), caspase-3/CPP32, and caspase-8/FLICE] and Fas-associated death domain protein (FADD) forming a death-inducing signalling complex (DISC) in Fas-mediated apoptosis of synoviocytes. METHODS: Synoviocytes were obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients. The number of dead cells was counted after treatment with anti-Fas monoclonal antibody in the presence of caspase-1-, -3-, or -8-specific inhibitors. The involvement of caspases and FADD in Fas-mediated apoptosis of RA synoviocytes was examined by immunoblot and immunoprecipitation analyses. RESULTS: RA synoviocytes expressed high levels of caspase-3, caspase-8, and FADD compared with OA synoviocytes. Interestingly, Fas ligation activated caspase-8 and caspase-3 with the cleavage of poly(ADP-ribose) polymerase (PARP), corresponding to apoptosis of RA synoviocytes. Furthermore, specific inhibitors for caspase-3 and caspase-8 but not caspase-1 suppressed Fas-induced apoptosis of RA synoviocytes in a dose- and time-dependent manner. Caspase-8-specific inhibitor suppressed the activation of caspase-3 after Fas ligation on RA synoviocytes. Importantly, FADD was selectively recruited to the Fas death domain during Fas-mediated apoptosis of RA synoviocytes, consistent with sensitivity to the Fas-mediated apoptosis. CONCLUSION: Our findings suggest that Fas-mediated apoptosis in synoviocytes may be regulated at the level of recruitment of FADD to the DISC, subsequently leading to the activation of the FADD/caspase-8/caspase-3 signalling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Synovial hyperplasia in HTLV-I associated arthropathy is induced by tumor necrosis factor-alpha produced by HTLV-I infected CD68+ cells.

OBJECTIVE: To investigate the pathogenic role of macrophage lineage (CD68+) cells in synovial proliferation in patients with human T cell leukemia virus I (HTLV-I) associated arthropathy (HAAP). METHODS: Synovial tissues obtained from 3 patients with HAAP and 3 patients with osteoarthritis (OA) were examined for the expression of tumor necrosis factor-alpha (TNF-alpha) mRNA, HTLV-I tax/rex mRNA, and number of CD68 by in situ reverse transcription assay and immunohistochemistry. Western blot and flow cytometric analyses were used to determine TNF-alpha production in HTLV-I infected synoviocytes. Changes in CD68+ cell population were examined by flow cytometric analysis. Proliferative effects of supernatants of HTLV-I infected synoviocytes on normal synoviocytes were also determined. RESULTS: TNF-alpha and HTLV-I tax/rex mRNA were preferentially expressed in CD68+ cells in HAAP synovia. Infection of OA synoviocytes by HTLV-I resulted in preferential expression in CD68+ cells, and these cells produced TNF-alpha. Supernatants of HTLV-I infected synoviocytes significantly enhanced the proliferation of normal synoviocytes through a TNF-alpha dependent pathway. CONCLUSION: Our results suggest HTLV-I viral tropism for CD68+ cells, and that HTLV-I infected synoviocytes were induced to produce TNF-alpha, which enhances synovial proliferation in HAAP.

Aged↗
PubMed1999

Tumor necrosis factor alpha regulation of the FAS-mediated apoptosis-signaling pathway in synovial cells.

OBJECTIVE: Fas-mediated apoptosis is observed in synoviocytes of patients with rheumatoid arthritis (RA), but not in those of patients with osteoarthritis (OA). The present study was conducted to elucidate the mechanisms that initiate induction of Fas-mediated apoptosis in RA synoviocytes. METHODS: Cultured OA synoviocytes, which are insensitive to Fas-mediated apoptosis in spite of Fas antigen expression, were used in these experiments. Synovial cell proliferation and cytotoxicity studies were performed using MTS and lactate dehydrogenase release assays. Surface expression of Fas antigen was analyzed by flow cytometry. The expression and function of apoptosis-signaling molecules, such as caspase 8 and caspase 3, were examined by immunoblot analysis. RESULTS: Tumor necrosis factor alpha (TNFalpha) induced proliferation of cultured OA synoviocytes. Fas ligation with anti-Fas monoclonal antibody (mAb) resulted in cytotoxic activity against cultured OA synoviocytes that had been pretreated with TNFalpha for 5 days, but not those pretreated for 2 days. In contrast, anti-Fas mAb did not show a cytotoxic effect against untreated cultured OA synoviocytes. A gradual up-regulation of caspase 8 and caspase 3, which played a role in the caspase cascade for Fas-mediated apoptosis, was observed in TNFalpha-treated cultured OA synoviocytes. In addition, Fas ligation to TNFalpha-treated cultured OA synoviocytes induced activation of caspase 8 and caspase 3, with subsequent cleavage of poly(ADP-ribose) polymerase (PARP), a substrate of activated caspase 3. More importantly, Z-IETD-FMK, a caspase 8 inhibitor, and Ac-DEVD-CHO, a caspase 3 inhibitor, almost completely inhibited Fas-mediated apoptosis of TNFalpha-treated cultured OA synoviocytes, whereas Ac-YVAD-CHO, a caspase 1 inhibitor, did not. CONCLUSION: Our results clearly demonstrate that TNFalpha stimulates synovial cells to proliferate as well as sensitizes the cells for Fas-mediated apoptosis, at least in part by up-regulation and activation of caspase 8 and caspase 3. These findings suggest that TNFalpha may be one of the factors providing sensitization of synovial cells to Fas-mediated apoptosis in RA.

Apoptosis↗
PubMed1999

Expression of murine HOXD9 during embryonic joint patterning and in human T lymphotropic virus type I tax transgenic mice with arthropathy resembling rheumatoid arthritis.

OBJECTIVE: To characterize the expression of murine HOXD9 during normal joint development and in arthritic joints of human T lymphotropic virus type I (HTLV-I) tax transgenic mice and the role of HTLV-I tax in HOXD9 expression. METHODS: Expression of HOXD9, HOXD1O, HOXD11, HOXD12, and HOXD13 genes in joint tissues at the ankle/foot regions of mouse embryos at day 10 to day 18 of gestation (E10-E18) and neonates within 10 days after birth was determined by reverse transcriptase-polymerase chain reaction and in situ reverse transcription methods. Adult synovial tissues from 5 HTLV-I tax transgenic mice with chronic polyarthritis and 4 nontransgenic (normal) mice were also examined for expression of these HOXD genes. The effect of HTLV-I on HOXD9 expression in cultured synoviocytes was studied by in vitro infection and transfection experiments. RESULTS: Expression of HOXD9 was detected in embryonic joints, preferentially on articular cartilage, only during the early stages of joint development (up to E15), whereas other HOXD genes were expressed throughout the embryonic and neonatal stages. In adult mice, transcripts of HOXD9 were specifically detected in synovial tissues from 4 of 5 arthritic mice, especially in the lining and sublining synovial cells, but not in synovial tissues of normal mice. Activation of HOXD9 was observed in cultured synoviocytes infected with HTLV-I in vitro as well as in those transfected with HTLV-I tax. CONCLUSION: Our findings suggest that HOXD9 is involved not only in the early stages of normal joint development, but may also be involved in the pathologic process of arthritis. HTLV-I tax appeared as an activator of this HOX gene in cultured synoviocytes.

Animals↗
PubMed1999

[Uric acid].

Explore the source record for details and available documents.

Female↗
PubMed1998

Evidence for autoantigens of Env/Tax proteins in human T cell leukemia virus type I Env-pX transgenic mice.

OBJECTIVE: To examine T cell clonotypes infiltrating into arthritic joints and to investigate whether human T cell leukemia virus type I (HTLV-I) env-pX gene products act as autoantigens in HTLV-I env-pX transgenic mice. METHODS: Complementary DNA (cDNA) encoding the V-D-J (third complementarity-determining region [CDR3]) region of T cell receptor beta chain was amplified by Vbeta family polymerase chain reaction. T cell clonotypes were detected by a single-strand conformational polymorphism method, and sequence analysis of the CDR3 region was performed. RESULTS: Distinct oligoclonal T cell expansion was observed in arthritic joints, and a conserved amino acid motif in the CDR3 region was found in T cells infiltrating joints. Moreover, several intraarticular T cells recognized HTLV-I Env and Tax proteins. CONCLUSION: Our results suggest that HTLV-I Env and Tax proteins act as autoantigens that are recognized by autoreactive T cells in inflamed arthritic lesions in the HTLV-I env-pX transgenic mouse. Thus, some T cells infiltrating the joint recognize Env or Tax protein. These cells may trigger chronic arthritis in HTLV-I env-pX transgenic mice.

Amino Acid Sequence↗
PubMed1998

Potential withdrawal of rheumatoid synovium by the induction of apoptosis using a novel in vivo model of rheumatoid arthritis.

OBJECTIVE: To investigate whether Fas-mediated apoptosis has potential as a new therapeutic strategy in rheumatoid arthritis (RA) by use of a novel model of RA in which human RA tissue is grafted into SCID mice. METHODS: Fresh rheumatoid synovial tissue including joint cartilage was grafted subcutaneously into the backs of SCID mice. Six weeks after engraftment, anti-Fas monoclonal antibody was injected intraperitoneally. Time-related apoptotic changes caused by anti-Fas monoclonal antibody in grafted synovium were evaluated by nick end-labeling histochemistry. RESULTS: Thirty-six hours after the injection, diffuse apoptotic changes were observed in the grafted synovia. Four weeks after the injection, rheumatoid synovial tissue diminished. CONCLUSION: This is the first report concerning the present effectiveness of anti-Fas monoclonal antibody in diminishing rheumatoid synovium in vivo, and suggests the possibility of a new strategy for treating rheumatoid arthritis by inducing Fas-mediated apoptosis.

Animals↗
PubMed1998

Monoclonal expansion of synoviocytes in rheumatoid arthritis.

OBJECTIVE: To examine whether synoviocytes from patients with rheumatoid arthritis (RA) have a stronger growth ability than those from patients with osteoarthritis (OA), and to determine whether these synoviocytes clonally expand in situ. METHODS: Synovial tissues from 13 RA patients and 4 OA patients were cultured, and their ability to form colonies in soft agarose was examined. RA and OA synoviocytes were also examined in varying concentrations of fetal calf serum (FCS)-containing medium to test the effects of FCS on colony formation. DNA was extracted from clones with colony-forming ability in nonpannus lesions and from synoviocytes in pannus lesions. Restriction fragment length polymorphism (RFLP) analysis was used to examine phosphoglycerate kinase 1 (PGK-1) gene patterns. Production of cytokines by these cells was also assessed. RESULTS: All 13 RA synoviocytes exhibited colony formation, whereas none of the 4 OA synoviocytes did. This tendency was also seen with all of the concentrations of FCS examined, although growth varied in a dose-dependent manner. In contrast to OA synovial clones, cloned RA synoviocytes obtained from colonies exhibited a partial RFLP PGK-1 gene pattern, suggesting that the clones originated from monoclonal cells. Of note, 3 of 7 noncloned synoviocytes from pannus lesions exhibited a monoclonal pattern. Pannus cells produced high levels of transforming growth factor beta and platelet-derived growth factor. CONCLUSION: These findings suggest that synoviocytes with a strong growth ability are present in the rheumatoid synovium, and that these cells expand monoclonally, particularly in pannus lesions.

Adult↗
PubMed1998

Extracellular human T cell leukemia virus type I tax protein stimulates the proliferation of human synovial cells.

OBJECTIVE: The present study was designed to investigate whether the proliferation of normal synovial cells from patients with meniscus injury is stimulated by human T cell leukemia virus type I (HTLV-I) Tax protein. METHODS: The effect of Tax protein on the proliferation of synovial cells was evaluated using a 3H-thymidine incorporation assay. Production of cytokines was determined by enzyme-linked immunosorbent assay. Nuclear factor kappaB (NF-kappaB) DNA binding activity and the transcription of several NF-kappaB-mediated genes was detected by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction. RESULTS: The proliferation of synovial cells, as well as their expression of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, and interleukin-6, was significantly enhanced by extracellular Tax at concentrations of 2.5 pM to 25 nM. In contrast, extracellular bacterial extract did not change the cytokine expression or the proliferation of these cells. Proliferation of synovial cells induced by Tax protein may be due to activated expression of several cytokines and protooncogenes that contain NF-kappaB regulatory sequences. CONCLUSION: Our results suggest that extracellular Tax can regulate the expression of endogenous cellular genes in synovial cells and may contribute to the NF-kappaB-mediated synovial hyperplasia.

Cell Division↗