Fujiko Tsukahara

Regulation of nuclear retention of glucocorticoid receptor by nuclear Hsp90

Heat shock protein 90 (Hsp90) has been demonstrated in both cytoplasm and nucleus, and regulates cytoplasmic retention of glucocorticoid receptor (GR). However, the role of nuclear Hsp90 in GR trafficking is less characterized. The present study examined the role of Hsp90 in nuclear retention of GR after ligand withdrawal. Hsp90 inhibitors; geldanamycin (GA) and radicicol (Rad), significantly accelerated nuclear export of GR after withdrawal of ligands including dexamethasone, corticosterone and RU486. GA accelerated relocalization of GR in the cytoplasm even when reimport of GR into the nucleus was inhibited by okadaic acid or when novel GR synthesis was inhibited by cycloheximide. Overexpression of wild type or nuclear-targeted Hsp90 attenuated Hsp90 inhibitor-induced acceleration of GR nuclear export, although nuclear Hsp90 showed higher activity than the wild type. Only nuclear-targeted Hsp90 prolonged basal nuclear retention of GR after withdrawal of dexamethasone and corticosterone. These results suggest that nuclear Hsp90 regulates the nuclear retention of GR.

Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin

In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT2A antagonist) and methysergide (5-HT1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine (NAN-190) (5-HT1A antagonist), but not by granisetron (5-HT3 antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors NG-nitro-Lrarginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor-α in brown adipocytes

Abstract We previously reported that tumor necrosis factor- α (TNF- α )/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF- α over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF- α on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF- α caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF- α (0.5–50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF- α on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, N G -nitro- l -arginine methyl ester ( l -NAME) and aminoguanidine, but not by d -NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3′,5′-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H -[1,2,4] oxadiazolo[4,3- a ]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF- α -suppressed lipoprotein lipase activity. These results suggest that TNF- α stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF- α on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells

Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell‐free condition and in an endothelial cell line (ECV304). In a cell‐free system, carvedilol attenuated NO‐dependent reduction of carboxy‐2‐phenyl‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl‐3‐oxide induced by a NO donor, 1‐hydroxy‐2‐oxo‐3‐(aminopropyl)‐3‐isopropyl‐1‐triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO‐saturated solution was attenuated by prior incubation with 0.1–10 μM carvedilol. NO‐induced fluorescence in 4,5‐diaminofluorescein‐2 diacethyl (DAF‐2DA)‐loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC50 of carvedilol for NOC5 or sodium nitroprusside‐induced fluorescence of DAF‐2DA in ECV304 cells was 1.0×10−7 M, which was similar to the reported IC50 of carvedilol for the antioxidant effect. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2‐2′(hydroxynitrosohydrazino)bis‐ethanamine was significantly attenuated by pretreatment with 1 μM carvedilol. Both free and cell‐associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.

Cationic amino acid transporter-2 mRNA induction by tumor necrosis factor-α in vascular endothelial cells

Nitric oxide (NO) synthesis may be coupled to the activity of the cellular L-arginine transporter, namely the cationic amino acid transporter. The present study examined tumor necrosis factor (TNF)-alpha-induced alterations in the gene expression of the cationic amino acid transporter (CAT) and NO production in human umbilical vein endothelial cells. In quiescent endothelial cells, CAT-1 mRNA expression, determined by reverse transcription-polymerase chain reaction, was dominant to that of CAT-2. TNF-alpha (10 ng/ml for 1-24 h) induced a time-dependent increase in CAT-2 but not CAT-1 expression. Moreover, TNF-alpha (1-30 ng/ml) treatment for 6 h induced a concentration-dependent increase in CAT-2 mRNA expression. The upregulation of CAT-2 expression by TNF-alpha was associated with enhanced nitrite accumulation in the culture medium (70% increase compared with vehicle-treated cells at 24 h). Thus, induction of the cationic amino acid transporter may constitute one mechanism for the TNF-alpha-induced NO production in human umbilical vein endothelial cells.

Possible involvement of L-arginine-nitric oxide pathway in modulating regional blood flow to brown adipose tissue of rats

To evaluate whether the l-arginine-nitric oxide (NO) pathway is involved in the regulation of regional blood flow to brown adipose tissue (BAT), the effects of two specific NO synthase inhibitors, NG-nitro-l-arginine methyl ester (l-NAME) and NG-monomethyl-l-arginine (l-NMMA), on the blood flow to interscapular brown adipose tissue (IBAT) were studied in urethane-anesthetized rats. Regional blood flow in MAT was measured with laser-Doppler flowmetry.An intravenous injection of l-NAME and l-NMMA, but not of either d-enantiomer, caused a transient and dose-dependent increase in IBAT blood flow. Dose-response curves for these NO synthase inhibitors showed that l-NAME was more potent than l-NMMA in increasing IBAT blood flow. We also observed a concomitant pressor effect accompanied by a slight decrease in heart rate following intravenous injection of l-NAME and l-NMMA. An elevation of IBAT blood flow and blood pressure induced by both l-NAME and l-NMMA was reversed by l-arginine in an enantiomerically specific manner. The increase in IBAT blood flow induced by NO synthase inhibitors was of shorter duration and less sensitive to l-arginine than the increase in blood pressure.Our results show that the WAY blood flow is increased by inhibition of NO synthase and that the response of IBAT vasculature to NO synthase inhibitors is different from that of the resistance vessels which regulate blood pressure. The involvement of l-arginine-NO pathways in modulating microcirculation in IBAT is suggested.

Tolerance to lipopolysaccharide-induced increase in vascular permeability in mouse skin

We investigated whether tolerance develops to the lipopolysaccharide-induced increase in vascular permeability of mouse skin on pretreatment with Salmonella typhimurium lipopolysaccharide. Lipopolysaccharide-induced plasma extravasation was assessed by determining Pontamine sky blue dye accumulation in the skin where lipopolysaccharide was injected s.c. 2 h previously. When mice were pretreated with lipopolysaccharide (0.15 mg/kg i.p.), the dye leakage induced by s.c. challenge with lipopolysaccharide (400 micrograms/site) was significantly, inhibited for 2-24 h after pretreatment, indicating the development of lipopolysaccharide tolerance. At 4 h after lipopolysaccharide (0.15 mg/kg i.p.), the dose-response curve of dye leakage against the challenge dose of lipopolysaccharide shifted about 2-fold to the higher dose. The dye leakage induced by lipopolysaccharide was inhibited by pretreatment with lipopolysaccharide in a dose-dependent manner (0.05-0.15 mg/kg i.p.). Lipopolysaccharide tolerance was not seen in adrenalectomized mice. When mice were pretreated with lipopolysaccharide and NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, at the same time, the hyporesponsiveness to lipopolysaccharide challenge disappeared. However, L-NAME was ineffective to inhibit the development of lipopolysaccharide tolerance when administered 24 h after lipopolysaccharide pretreatment or just before the lipopolysaccharide challenge. Tumor necrosis factor-alpha and interleukin-1 alpha but not interleukin-6 induced a similar hyporesponsiveness to lipopolysaccharide. These results suggest that tolerance develops to the lipopolysaccharide-induced increase in vascular permeability in mouse skin after a single lipopolysaccharide administration and that endogenous glucocorticoids and NO are necessary for induction of lipopolysaccharide tolerance. Hyporesponsiveness induced by lipopolysaccharide pretreatment may be mediated by production of some cytokines such as tumor necrosis factor-alpha or interleukin-1 alpha.

Effect of reserpine pretreatment on brown fat iodothyronine 5'-deiodinase of mouse

Abstract— The effect of pretreatment with reserpine (1 mg kg−1 i.p. daily for 7 days) on the regulation of iodothyronine 5′‐deiodinase (5′D) in mouse brown adipose tissue (BAT) has been examined. 5′D activity of BAT homogenate was assessed by the in‐vitro formation of 3,5,3′‐triiodothyronine from thyroxine and 3,3′‐diiodothyronine from 3,3′,5′‐triiodothyronine in the presence of 20 mM dithiothreitol. Reserpine treatment decreased the stimulation of BAT 5′D induced by acute cold exposure (4°C, 2h) without a significant decrease in the basal 5′D activity, whereas stimulation of BAT 5′D elicited by noradrenaline (0.4 and 0.8 mg kg−1 s.c. 2 h previously) was not augmented after reserpine treatment. Although both noradrenaline and acute cold exposure increase BAT 5′D through α1‐adrenoceptors, our results show that chronic reserpine treatment prevents the effect of cold, but does not induce α1‐adrenoceptor supersensitivity in BAT.

Paradoxical counteraction by imatinib against cell death in myeloid progenitor 32D cells expressing p210BCR-ABL

Chronic myeloid leukemia (CML) is believed to be caused by the tyrosine kinase p210BCR-ABL, which exhibits growth-promoting and anti-apoptotic activities. However, mechanisms that allow cell differentiation in CML still remain elusive. Here we established tetracycline (Tet)-regulatable p210BCR-ABL-expressing murine 32D myeloid progenitor (32D/TetOff-p210) cells to explore p210BCR-ABL-induced cell death and differentiation. Tet-regulatable overexpression of p210BCR-ABL induced cell death due to the activation of both caspase-1 and caspase-3, coincident with the differentiation from myeloid progenitors into CD11b+Ly6C+Ly6G+ cells with segmented nuclei, exemplified as granulocytic myeloid-derived suppressor cells (G-MDSC), and the ability to secrete IL-1β, TNF-α, and S100A8/A9 into the culture supernatant. Treatment with imatinib almost completely abrogated all these phenotypes. Moreover, overexpression of a sensor of activated caspase-1 based on fluorescence resonance energy transfer (FRET) probe enabled us to detect activation of caspase-1 in a human CML cell line, K562. Furthermore, increased numbers of splenic G-MDSC associated with enhancement of S100A8/A9 production were observed in transgenic mice expressing p210BCR-ABL compared with that in wild-type mice. We also propose the novel mode of cell death in this 32D/TetOff-p210 system termed as myeloptosis.

C1D is not directly involved in the repair of UV-damaged DNA but protects cells from oxidative stress by regulating gene expressions in human cell lines

&NA; A small nuclear protein, C1D, has roles in various cellular processes, transcription regulation, genome stability surveillance, DNA repair and RNA processing, all of which are required to maintain the host life cycles. In the previous report, C1D directly interacts with XPB, a component of the nucleotide excision repair complex, and C1D knockdown reduced cell survival of 27–1 cells, CHO derivative cells, after UV irradiation. To find out the role of C1D in UV‐damaged cells, we used human cell lines with siRNA or shRNA to knockdown C1D. C1D knockdown reduced cell survival rates of LU99 and 786‐O after UV irradiation, although C1D knockdown did not affect the efficiency of the nucleotide excision repair. Immunostaining data support that C1D is not directly involved in the DNA repair process in UV‐damaged cells. However, H2O2 treatment reduced cell viability in LU99 and 786‐O cells. We also found that C1D knockdown upregulated DDIT3 expression in LU99 cells and downregulated APEX1 in 786‐O cells, suggesting that C1D functions as a co‐repressor/activator. The data accounts for the reduction of cell survival rates upon UV irradiation.