Etsushi Fukawa

Thrombin-induced expression of RANTES mRNA through protease activated receptor-1 in human synovial fibroblasts

Objective: To examine the effects of thrombin on RANTES mRNA expression through protease activated receptor in synovial fibroblasts in patients with rheumatoid arthritis (RA). Methods: A semiquantitative reverse transcriptase-polymerase chain reaction and reporter gene assay were performed using cultured human synovial fibroblasts from patients with RA. The up regulatory effects of thrombin on RANTES mRNA expression were tested. In addition, the roles of protease activated receptors (PARs) were analysed. Results: PAR-1 and PAR-3, but not PAR-4, were expressed in synovial fibroblasts. Thrombin induced RANTES mRNA expression in a time dependent manner in synovial fibroblasts expressing PAR-1. A reporter gene assay showed that thrombin-induced RANTES gene expression was through PAR-1, but not PAR-3. Conclusions: Thrombin induced RANTES mRNA expression through a PAR-1 mediated pathway, possibly indicating that thrombin has an important role in migration of inflammatory cells by RANTES to the synovium in patients with RA.

Tumor necrosis factor α (TNF-α)-induced RANTES chemokine expression via activation of NF-κB and p38 MAP kinase: roles of TNF-α in alcoholic liver diseases

Abstract Background/Aims : Increased concentration of plasma tumor necrosis factor α (TNF-α) correlates with the clinical course of alcoholic liver diseases. In addition, hepatic RANTES which migrates CD4 T lymphocytes to liver is increased in patients with alcoholic hepatitis. We investigated that roles of TNF-α on RANTES expression in hepatocytes. Methods : HLE cells were treated with TNF-α in the presence, or absence of several inhibitors. Enzyme-linked immunoassay and reverse transcriptase-polymerase chain reaction were performed for the measurement of protein production and mRNA of RANTES, respectively. Moreover, DNA-binding activity of NF-κB was investigated using electrophoretic mobility shift assay. To examine effects of TNF-α on RANTES gene expression, luciferase assay was performed. Results : TNF-α clearly up-regulated RANTES expression in a time-dependent fashion and induced DNA-binding activity of NF-κB. Moreover, TNF-α-induced RANTES expression was completely inhibited by SB203580, but not calphostin C and wortmannin. Luciferase assay showed that TNF-α increased RANTES gene expression and mutation of NF-κB binding sites in the RANTES promoter ablated TNF-α inducibility. Conclusions : We showed that RANTES was transcriptionally induced in human hepatoma cells by treatment with TNF-α via activation of NF-κB and p38 MAP kinase, presumably suggesting that TNF-α-induced expression of RANTES plays important roles in cell-mediated liver injury in alcoholic liver diseases.

Relative glucocorticoid potency revisited.

To determine the relative potency of synthetic glucocorticoids, glucocorticoid receptor expressing cells were transfected with a hormone-inducible reporter gene, and were cultured in the presence of various glucocorticoid ligands. Hormonal inducbility was determined by means of a chloramphenicol acetyltransferase assay. Dexamethasone and prednisolone, as well as cortisol, induced the expression of the reporter gene in a dose-dependent fashion. The relative potency of each ligand was in this order when inducibility was quantitatively assessed. In conclusion, the transcription assay described here may be a convenient and alternative method to evaluate the relative potency of given glucocorticoids.

Natural course of diabetic peripheral neuropathy in spontaneous-onset diabetic Chinese hamsters

We investigated metabolic and pathological changes in the peripheral nerve of the spontaneous-onset diabetic Chinese hamster. Electrophysiological examination revealed that the motor nerve conduction velocity was significantly decreased at 10 months and afterwards, however, the F-wave latency was significantly increased at 5 months and afterwards. Concerning sciatic nerve contents of sorbitol, myo- and scyllo-inositol, the content of sorbitol was not significantly increased at 5 months, but, myo- and scyllo-inositol were significantly decreased at 5 months and thereafter. At 10 and 15 months, however, sciatic nerve content of sorbitol was significantly increased. On morphological examination, loss of large myelinated fiber and reciprocal increase in degenerative fiber were also seen in sciatic nerve, but not in tibial nerve, at 5 months. At 15 months, these morphological changes were also found in the tibial as well as the sciatic nerve. Thus, we may hypothesize that F-wave latency is useful in the detection of initial diabetic neuropathy, and that the initial pathological changes in diabetic neuropathy of diabetic Chinese hamsters are predominantly found in the proximal site of peripheral nerves.

Nuclear factor-κB regulates RANTES chemokine expression in response to tumor necrosis factor-α in fibroblast-like synoviocytes

Abstract We investigated the role of nuclear factor (NF)-κB on tumor necrosis factor (TNF)-α-induced regulated upon activation, normal T-cell expressed and secreted (RANTES) expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis (RA). Using cultured human fibroblast-like synoviocytes from patients with RA, semiquantitative reverse transcriptase-polymerase chain reaction, electrophoretic mobility shift assay, and Western blot were performed for RANTES expression, NF-κB activation, and degradation of IκB, respectively. In addition, the transcriptional effect of TNF-α on RANTES gene expression was analyzed by reporter gene assay. We found that TNF-α clearly induced RANTES protein production and expression of RANTES mRNA in a time-dependent manner. Furthermore, TNF-α persistently induced NF-κB activation caused by IκBα and IκBβ1 degradation. Supershift analysis revealed that TNF-α-induced DNA-binding complexes were composed principally of the p65 and p50 Rel family members. Moreover, transcriptional activation of the RANTES promoter by TNF-α was dependent on specific NF-κB response elements that were regulated by NF-κB. Results herein indicate that NF-κB activation caused by degradation of IκBα and IκBβ1 by TNF-α increased RANTES gene expression in fibroblast-like synoviocytes, suggesting that NF-κB plays an important role in the migration of inflammatory cells by RANTES to the synovium in patients with rheumatoid arthritis.