Fuminori Hirano

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.

Chenodeoxycholic acid and taurochenodexycholic acid induce anti‐apoptotic cIAP‐1 expression in human hepatocytes

Background and Aims:  Increased concentration of endogenous bile acids in the liver correlates with clinical features of cholestatic liver diseases. Recently, it was reported that non‐toxic hydrophobic bile acid activated a survival signaling pathway via phosphatidylinositol 3 (PI3) kinase in hepatocytes. However, whether bile acid induces inhibitors of apoptosis protein (IAPs) directly in human hepatocytes remains unknown. This study investigated effects of bile acids on cIAP‐1, cIAP‐2 and XIAP expression in hepatocytes.

Inhibition of NF-κB-dependent transcription of human immunodeficiency virus 1 promoter by a phosphodiester compound of vitamin C and vitamin E, EPC-K1

We investigated the effect of EPC-K1, which is a phosphodiester compound of vitamin E and vitamin C, on NF-kappaB activity in human cultured astrocytoma cells T98G. In TNFalpha-stimulated T98G cells, treatment with EPC-K1 inhibited both DNA binding activity and transactivation of NF-kappaB in a dose-dependent manner, and the suppressive effect of EPC-K1 was stronger than either that of vitamin E or vitamin C. Moreover, we showed that in TNFalpha-stimulated T98G cells treatment with EPC-K1 repressed NF-kappaB-dependent activation of the human immunodeficiency virus 1 promoter. In contrast, TNFalpha-induced activation of the human immunodeficiency virus 1 promoter was not completely inhibited by either treatment with vitamin E or vitamin C. We, thus, suggest that EPC-K1 is considered to be one of the inhibitory agents of NF-kappaB.

The inhibitory effect of bisphosphonates on glucocorticoid‐induced RANKL expression in human cells

Objective: RANKL is known to play an important role in activating osteoclasts and advancing the progress of osteoporosis. However, little is known about the effect of bisphosphonates on glucocorticoid‐induced RANKL expression in human cells. Our study was intended to clarify effects of bisphosphonates on glucocorticoid‐induced RANKL expression in human cells. Methods: Human T lymphoblastic cell line Jurkat and human osteosarcoma cell line MG‐63 were used for the following experiments. RANKL expression in two cell lines was measured using reverse transcription polymerase chain reaction (RT‐PCR) analysis and enzyme immunoassay (EIA). Luciferase assays using pGRE‐Luc were also performed. Results: In Jurkat and MG‐63 cells, dexamethasone induced expression of soluble RANKL (sRANKL) protein in supernatants and RANKL mRNA in cells. Moreover, bisphosphonates, but not cyclooxygenase inhibitors, repressed dexamethasone‐induced sRANKL protein production. By contrast, glucocorticoid receptor‐driven transcriptional activity was not inhibited by bisphosphonates. Conclusion: Glucocorticoid induced RANKL expression in human cells derived from T lymphocytes and osteoblasts. Bisphosphonates inhibited glucocorticoid‐induced RANKL expression, suggesting that these effects might be a new therapeutic mechanism for bisphosphonates.

Fibrates suppress chenodeoxycholic acid-induced RANTES expression through inhibition of NF-κB activation

Fibrates, hypolipidemic agents, are reported to be effective in treatment of primary biliary cirrhosis. However, the mechanism involved in therapeutic benefits of fibrates in primary biliary cirrhosis remains unknown. In contrast, hepatic regulated upon activation, normal T-cell expressed and secreted (RANTES) is increased in patients with primary biliary cirrhosis and bile acids up-regulate RANTES expression in hepatocytes. The role of fibrates in bile acid-induced RANTES expression was investigated in human hepatoma cells; 100 microM of bezafibrate and fenofibrate decreased expression of chenodeoxycholic acid-induced RANTES mRNA and protein. In addition, luciferase enzyme assay using RANTES promoter-luciferase reporter plasmid revealed that 100 microM of bezafibrate and fenofibrate transcriptionally reduced chenodeoxycholic acid-induced RANTES gene expression. Moreover, bezafibrate clearly repressed DNA-binding activity of nuclear factor-kappaB (NF-kappaB) induced by chenodeoxycholic acid. Therefore, fibrates might be inhibitory agents of inflammatory cell migration by RANTES to the liver in patients with primary biliary cirrhosis, possibly indicating that fibrates are therapeutic agents in primary biliary cirrhosis.

Effects of ursodeoxycholic acid and chenodeoxycholic acid on major histocompatibility complex class I gene expression

We investigated the effect of ursodeoxycholic acid on major histocompatibility complex class I gene expression in cultured human hepatoma cells. Ursodeoxycholic acid, which is now being used for the treatment of various autoimmune liver diseases, paradoxically increased the mRNA level of major histocompatibility complex class I. However, endogenous bile acids, for example, chenodeoxycholic acid, increased major histocompatibility complex class I mRNA expression more strongly compared with ursodeoxycholic acid. Concerning the interplay between ursodeoxycholic and chenodeoxycholic acids, these bile acids additively induced major histocompatibility complex class I mRNA expression. In contrast, when the total concentration of ursodeoxycholic and chenodeoxycholic acids was kept constant, the expression of major histocompatibility complex class I mRNA appeared to decrease in a dose-dependent manner with an increasing ratio of ursodeoxycholic acid. These findings indicate that the beneficial action of ursodeoxycholic acid may be related to this relative decrease in major histocompatibility complex class I gene expression.

Homeobox gene CDX2 inhibits human pancreatic cancer cell proliferation by down-regulating cyclin D1 transcriptional activity.

Objectives: Homeobox gene caudal related homeobox gene 2 (CDX2) is an intestine-specific tumor suppressor gene. This study is intended to investigate the effect of CDX2 expression on cell proliferation and cyclin D1 expression in pancreatic cancer cells. Methods: Four pancreatic ductal adenocarcinoma cell lines (PancQGO-1, BxPC-3, MIAPaCa-2, CFPAC-1), 1 islet carcinoma cell line (QGP-1), and 1 adenosquamous carcinoma cell line (KP-3) were analyzed for CDX1 and CDX2 expression using real-time reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of pancreatic cancer cells was analyzed using WST-1 assay after CDX2 transfection. Luciferase assay was performed to examine the effects of CDX2 on cyclin D1 transcriptional activity. Results: CDX2 was expressed at a significantly higher level in QGP-1 cells than in KP-3 cells. Moreover, CDX2 was expressed at a middle level in 4 pancreatic ductal adenocarcinoma cells. Cell proliferation and cyclin D1 mRNA level were inhibited significantly after CDX2 transfection in pancreatic cancer cells. Furthermore, CDX2 inhibited exogenous nuclear factor &kgr;B-p65-induced luciferase gene expression in a dose-dependent manner. In addition, CDX2 inhibited pGL2HIVD1&kgr;B2-luciferase activity. Conclusions: CDX2 might play a role in inhibiting cell proliferation and repressing cyclin D1 transcriptional activity through the proximal nuclear factor &kgr;B binding site in pancreatic cancer cells.

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.

Breast arterial calcification and hypertension associated with vertebral fracture

Aim:  Arterial calcification and osteoporosis commonly accompany one another in postmenopausal women. Hypertension is a known contributing factor to arterial calcification. Thus, we aimed to investigate any associations between hypertension, arterial calcification and vertebral fractures in a cross‐sectional study in Japanese postmenopausal women.