Han-Jung Chae

ESTROGEN REGULATES CYTOKINE RELEASE IN HUMAN MAST CELLS

Estrogens are important for bone homeostasis and are classified as anti-resorptive agents. In ovariectomized rats, mast cell changes occurred during the activation of resorption. In addition, quantitative changes occurred in mast cell population residing near the site undergoing resorption. Considering these studies, mast cells may play a role in osteoporosis. Therefore, it is of paramount importance to study mast cell cytokine production also in the presence or absence of estrogen. When cultured in the absence of estrogen, human mast cells treated with PMA or A23187 demonstrated significantly greater release of TNF-α and IL-6 than cells grown under estrogen-depleted condition. Our results show that treatment of mast cells with estrogen prevented PMA or A23187-stimulated TNF-α or IL-6 release. These data provide evidence for a potent inhibition of cytokines by estrogen in human mast cells. This study may help to explain the association between mast cells and osteoporosis.

Nitric oxide production by high molecular weight water-soluble chitosan via nuclear factor-κB activation

Abstract High molecular weight water-soluble chitosan (WSC), having an average molecular weight of 300u2008000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the effect of WSC on the production of nitric oxide (NO) in RAW 264.7 macrophages was evaluated. Water-insoluble chitosan alone has been previously shown to exhibit in vitro stimulatory effect on macrophages NO production. However, WSC had no effect on NO production by itself. When WSC was used in combination with recombinant interferon-γ (rIFN-γ), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of WSC on NO synthesis was shown 24 h after treatment with rIFN-γ. The increased production of NO from rIFN-γ plus WSC-stimulated RAW 264.7 macrophages was decreased by the treatment with N G -monomethyl- l -arginine ( N G MMA). The increase in NO synthesis was reflected, as an increased amounts of inducible NO synthase protein. In addition, synergy between rIFN-γ and WSC was mainly dependent on WSC-induced tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) activation. The present results indicate that the capacity of WSC to increase NO production from rIFN-γ-primed RAW 264.7 macrophages is the result of WSC-induced TNF-α secretion via the signal transduction pathway of NF-κB activation.

Hypoxia induces apoptosis by caspase activation accompanying cytochrome C release from mitochondria in MC3T3E1 osteoblasts. p38 MAPK is related in hypoxia-induced apoptosis.

The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, western blot analysis was performed using anticaspase-3 or -6 antibody. The 10-kDa protein, corresponding to the active products of caspase-3 and the 10-kDA protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which processing of the full length form of caspase-3 and -6 was evident. With a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we have observed that pre-treatment with SB203580, a selective p38 MAP kinase (p38 MAPK) inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAP kinase -JNK/SAPK or ERK- was not or modestly activated. These results suggest that p38 MAPK positively regulates hypoxia-induced apoptosis in MC3T3E1 osteoblasts.

Signal transduction of thapsigargin-induced apoptosis in osteoblast

The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+-ATPase, was investigated in osteoblasts. We induced apoptosis in murine osteoblastic MC3T3E1 cells by exposure to the thapsigargin. Thapsigargin transiently increased the phosphotransferase activity of c-Jun N-terminal kinases1 (JNK1), which might in turn activate transcriptional activity of activation protein-1 (AP-1). We then prepared extracts from thapsigargin-treated MC3T3E1 cells and monitored cleavage of acetyl-YVAD-AMC and acetyl-DEVD-AMC, fluorogenic substrates for caspase 1-like and caspase 3-like proteases, respectively. Thapsigargin significantly increased the proteolytic activity of caspase 3-like proteases, but not the activity of caspase 1-like proteases. Furthermore, thapsigargin increased the transcriptional activity of nuclear factor-kappaB (NF-kappaB). These data suggest that thapsigargin-induced apoptosis in osteoblasts may be via activation of JNK1, caspase 3-like family proteases, and transcriptional factors including AP-1 and NF-kappaB.

Bax inhibitor-1 regulates the expression of P450 2E1 through enhanced lysosome activity.

In this study, we explored the role of Bax inhibitor-1 (BI-1) on the expression of P450 2E1 and related ROS production. P450 2E1 protein, not mRNA, was expressed at relatively low levels in BI-1 plasmid-transfected cells (BI-1 cells) compared with neomycin-resistant vector-transfected cells (Neo cells). When exposed to ER stress, P450 2E1 expression and activity and ER membrane lipid peroxidation increased in both Neo cells and BI-1 cells, but to a lesser degree in BI-1 cells. This observation correlated with the lower level of ER stress in BI-1 cells than Neo cells. To examine the BI-1-associated P450 2E1 degradation mechanism, cells were treated with the lysosome inhibitor, bafilomycin and the proteasome inhibitor, MG132. Bafilomycin recovered the reduced P450 2E1 expression in BI-1 cells, but did not affect P450 2E1 expression in Neo cells. Next, proteosomal and lysosomal activities in Neo cells were compared to those in BI-1 cells. Although proteosomal activity was similar between Neo and BI-1 cells, LysoTracker and acridine orange labeling, lysosomal V-ATPase activity, and lysosomal cathepsin B expression were higher in BI-1 cells than in Neo cells. In the presence of ER stress, lysosomal activities decreased in Neo cells but did not change in BI-1 cells. P450 2E1 expression and ER membrane lipid peroxidation were greater in the hepatocytes and livers of BI-1 knock-out mice than in BI-1 wild-type cells and mice. Our results suggest that the BI-1-mediated enhancement of lysosomal activity regulates P450 2E1 expression and resultant ROS accumulation.

Inhibitory effect of anaphylactic shock by caffeine in rats

Caffeine is known to reduce evoked histamine secretion, but the effects of caffeine on anaphylactic shock have not been clarified. We have investigated the effects of caffeine on anaphylactic shock in rats. Systemic anaphylactic shock by compound 48/80 injection was monitored for 1 h. An IgE-dependent local anaphylactic shock was generated by sensitizing the skin with anti-dinitrophenyl (DNP) IgE followed 48 h later with an injection of antigen. Caffeine inhibited compound 48/80-induced anaphylatic shock to 40% with a dose of 1 mg/kg. Caffeine (0.1 mg/kg) inhibited to 56.4+/-0.4% passive cutaneous anaphylactic shock activated by anti-DNP IgE. Caffeine (5-20 mM) significantly inhibited histamine release from rat peritoneal mast cells (RPMCs) activated by compound 48/80 or anti-DNP IgE. Especially, caffeine (20 mM) inhibited by 96.7+/-0.5% histamine release activated by compound 48/80. Moreover, caffeine (1-20 mM) had a significant inhibitory effect on anti-DNP IgE-induced tumor necrosis factor-alpha production from RPMCs. The level of cAMP in RPMCs, when caffeine (20 mM) was added, increased significantly after 5-60 min compared with that of a normal control. These results indicate that caffeine inhibits immediate-type allergic reactions by inhibition of mast cell degranulation in vivo and in vitro.

Inhibitory effect of sodium salicylate on nitric oxide production from TM4 sertoli cells.

Nitric oxide (NO) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the production of NO in TM4 Sertoli cells. TM4 Sertoli cells produced a small amount of NO upon treatment with recombinant interferon-gamma (rIFN-gamma). The effect of rIFN-gamma was enhanced markedly by the addition of recombinant TNF-alpha (rTNF-alpha) in a dose-dependent manner. NaSal (10 and 20 mM) significantly inhibited NO production from TM4 Sertoli cells induced by rIFN-gamma plus rTNF-alpha. In addition, rIFN-gamma in combination with rTNF-alpha showed a marked increase of the expression of inducible NO synthase (iNOS) protein. Western blot analysis revealed that NaSal (10 and 20 mM) blocked a step of iNOS protein synthesis. The rIFN-gamma plus rTNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation was significantly blocked by NaSal (10 and 20 mM). On the other hand, neither staurosporine nor polymyxin B significantly inhibited NO production from TM4 Sertoli cells induced by rIFN-gamma plus rTNF-alpha. The present results indicate that NaSal inhibits rIFN-gamma plus rTNF-alpha-induced NO production in TM4 Sertoli cells via the signal transduction pathway of NF-kappaB activation.

Nitric oxide prevents the IFN-γ/LPS-induced hepatotoxicity in a protein kinase G-independent manner

Although it has been well known that the role of LPS on hepatotoxicity is mediated through TNF-α, the direct cytotoxic effect of LPS on IFN-γ-primed hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that the IFN-γ-mediated death of murine embryonic liver BNL CL2 cells is potentiated by LPS (0.5 μg/ml). In addition, an exogenous NO donor, sodium nitroprusside (SNP) significantly prevents cell death induced by IFN-γ alone or IFN-γ plus LPS (IFN-γ/LPS) in a dose-dependent manner over 25 μM. SNP significantly blocked the death of BNL CL2 cells only when it was added within 12 hr after treatment of IFN-γ and IFN-γ/LPS. The preventive effect of SNP occurred in parallel with the suppression of caspase 3-like protease activation. We have also demonstrated that a relatively high concentration as well as an appropriate period of exposure to NO may be critical to maintain cell viability from the cytotoxic effect of IFN-γ and IFN-γ/LPS. Furthermore, the preventive effect of SNP on IFN-γ/LPS-induced cell death is mediated by a protein kinase G (PKG)-independent manner.