Novel anti-apoptotic mechanism of A20 through targeting ASK1 to suppress TNF-induced JNK activation.

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of nuclear factor-κB (NF-κB) activation and apoptosis in tumor necrosis factor (TNF) receptor 1 signaling pathway. Although the molecular basis for the anti-NF-κB function of A20 has been well elucidated, the anti-apoptotic function of A20 is largely unknown. Here, we report a novel mechanism underlying the anti-apoptotic function of A20: A20 blocks TNF-induced apoptosis through suppression of c-jun N-terminal kinase (JNK) by targeting apoptosis signal-regulating kinase1 (ASK1). First, the ectopic expression of A20 drastically inhibits TNF-induced JNK activation and apoptosis in multiple cell types including those deficient of NF-κB activation. Unexpectedly, the blunting effect of A20 on TNF-induced JNK activation is not mediated by affecting the TNFR1 signaling complex formation. Instead, A20 interacts with ASK1, an important MAPKK kinase in the JNK signaling cascade. More importantly, overexpression of wild-type A20, but not of mutant A20 (ZnF4; C624A, C627A), promotes degradation of the ASK1 through the ubiquitin-proteasome system. Taken together, the results from this study reveal a novel anti-apoptotic mechanism of A20 in TNF signaling pathway: A20 binds to ASK1 and mediates ASK1 degradation, leading to suppression of JNK activation and eventually blockage of apoptosis.

Caspase-8 has an essential role in resveratrol-induced apoptosis of rheumatoid fibroblast-like synoviocytes.

OBJECTIVE: Resveratrol is a naturally occurring polyphenol, which possesses chemotherapeutic potential through its ability to trigger apoptosis. The objective of this study was to investigate the major determinant for the apoptotic cell death induction by resveratrol in fibroblast-like synoviocytes (FLS) derived from patients with RA. METHODS: The effect of resveratrol on apoptotic cell death was quantified in a population of subG1 in RA FLS by flow cytometry. The underlying signalling mechanism for apoptotic death was examined by analysing mitochondrial membrane potential, activation of the caspase cascade and translocation of Bid. RESULTS: We show that activation of caspase-8 is essential for triggering resveratrol-induced apoptotic signalling via the involvement of the mitochondrial pathway in RA FLS. Our findings also suggest that this enhanced apoptosis caused by resveratrol occurred in RA FLS irrespective of p53 status. Exposure to resveratrol caused extensive apoptotic cell death, along with a caspase-dependent (activation of caspase-9 and -3, poly ADPribose polymerase (PARP) cleavage and mitochondrial cytochrome c release) or caspase-independent [translocation of apoptosis-inducing factor (AIF) to the nucleus] signalling pathway. Analysis of upstream signalling events affected by resveratrol revealed that the activated caspase-8 triggered mitochondrial apoptotic events by inducing Bid cleavage without any alteration in the levels of Bax, Bcl-xL or Bcl2. The caspase-8 inhibitor or over-expression of crmA abrogated cell death induced by resveratrol and prevented processing of the downstream cascade. CONCLUSION: The results suggest that resveratrol causes activation of caspase-8, which in turn results in modulation of mitochondrial apoptotic machinery to promote apoptosis of RA FLS.

Expression profiling of radiation-induced genes in radiodermatitis of hairless mice.

BACKGROUND: Radiation induces many cellular events leading to radiodermatitis. OBJECTIVES: The aim of this study was to establish a radiodermatitis model using experimental animals, and to examine the expression profile of radiation-induced genes. METHODS: Hairless mice were irradiated on the dorsal skin; then total RNAs were isolated and microarray hybridizations were performed. RESULTS: Irradiation with a total of 40 Gy (10 Gy day-1 for four consecutive days) provokes radiodermatitis in the hairless mouse. After microarray analysis, 130 genes that showed upregulation by radiation were selected and organized into four different clusters, depending on the time-kinetic pattern. Classification of these genes into several functional categories revealed that various biological processes were globally affected by radiation. These include transcription regulation, signal transduction, cell communication, cell death regulation and metabolism. CONCLUSIONS: These results demonstrate the complexity of the transcriptional profile of the radiation response, providing important clues on which to base further investigations of the molecular events underlying radiodermatitis.

Serum after partial hepatectomy stimulates iNOS gene transcription via downstream NF-kappa B site.

It has been known that the expression of inducible nitric oxide synthase (iNOS) is up-regulated during hepatic regeneration. The present study characterized the molecular mechanisms involved in the transcriptional activation of iNOS gene by using the serum after partial hepatectomy (post-PH serum) in vitro. The post-PH serum rapidly induced iNOS mRNA expression, which was blocked by anti-tumor necrosis factor-alpha (TNF-alpha) antibody in BNL CL.2 cells, murine embryonic liver cell line. In addition, EMSAs using a NF-kappa B-specific oligomer showed that the up-regulated iNOS mRNA expression in cells treated with post-PH serum correlated with transient activation of NF-kappa B complex (p50/p65 heterodimer). Transient transfection of BNL CL.2 cells with iNOS promoter linked to a CAT reporter gene showed the transcriptional activation of iNOS promoter by post-PH serum. Furthermore, site-directed mutational analysis of the two NF-kappa B sites individually or in combination revealed that iNOS expression by post-PH serum is regulated by the downstream NF-kappa B site, but not by upstream NF-kappa B site. Taken together, these results suggest that the downstream NF-kappa B site acts as an essential component for the iNOS expression by post-PH serum during hepatic regeneration.

Enhancement of lysophosphatidic acid-induced ERK phosphorylation by phospholipase D1 via the formation of phosphatidic acid.

We made stable cell lines overexpressing PLD1 (GP-PLD1) from GP+envAm12 cell, a derivative of NIH 3T3 cell. PLD1 activity and extracellular signal-regulated kinase (ERK) phosphorylation were enhanced in GP-PLD1 cells by the treatment of lysophosphatidic acid (LPA). In contrast, these LPA-induced effects were attenuated with the pretreatment of pertussis toxin (PTX) or protein kinase C (PKC) inhibitor. Moreover, accumulation of phosphatidic acid (PA), a product of PLD action, potentiated the LPA-induced ERK activation in GP-PLD1 cells while blocking of PA production with the treatment of 1-butanol attenuated LPA-induced ERK phosphorylation. From these results, we suggest that LPA activate PLD1 through pertussis toxin-sensitive G protein and PKC-dependent pathways, then PA produced from PLD1 activation facilitate ERK phosphorylation.

Alpha1-adrenoceptors involvement in painful diabetic neuropathy: a role in allodynia.

To investigate whether alpha1-adrenoceptors are involved in pain behaviors in streptozotocin (STZ)-induced diabetic rats, we measured the effects of phenylephrine or prazosin on allodynia in the diabetic rats. Phenylephrine aggravated allodynia, while prazosin alleviated allodynia in the diabetic rats. We also measured alpha1-adrenoceptors gene expression or density of [3H]-prazosin binding sites in the dorsal root ganglia (DRG) and spinal cord in painful diabetic rats. Alpha1-adrenoceptors mRNA and density of [3H]prazosin binding sites were increased in the DRG of the diabetic rats, however there were no significant differences in alpha1-adrenoceptors expression in the spinal cord between the control and diabetic rats. These results suggest increased alpha1-adrenoceptors in the DRG may play a role in the pathogenesis of painful diabetic neuropathy.

Acetaminophen inhibits iNOS gene expression in RAW 264.7 macrophages: differential regulation of NF-kappaB by acetaminophen and salicylates.

Acetaminophen is a widely used analgesic and anti-inflammatory drug that is considered a good alternative to salicylates for individuals who cannot tolerate salicylates. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. Recent evidence suggests that anti-inflammatory effect of salicylates lies in the inhibition of iNOS, but nothing has been reported about the direct effect of iNOS expression by acetaminophen. The present study was designed to elucidate sequentially the action mechanisms of acetaminophen and salicylates (aspirin and sodium salicylate) on lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-induced iNOS expression in RAW 264.7 macrophages. Both acetaminophen and salicylates inhibited NO production and iNOS protein expression in a dose dependent manner. Acetaminophen inhibited iNOS mRNA expression, promoter activity of iNOS gene and nuclear factor-kappa B (NF-kappaB) binding activity induced by LPS plus IFN-gamma, whereas salicylates did not show any effect on them. In addition, salicylates did not affect on iNOS mRNA stability induced by LPS plus IFN-gamma. Furthermore, the inhibition of iNOS protein expression and NO production by salicylates was disappeared when salicylates were added for only 5 h to inhibit the early event of iNOS expression. Aspirin also dose dependently inhibited iNOS enzyme activity in cell-free extracts, whereas no significant differences were observed in extracts treated with sodium salicylate or acetaminophen. These findings suggest that acetaminophen may exert analgesic or anti-inflammatory effect by inhibiting iNOS expression induced by LPS plus IFN-gamma at transcriptional level by suppression of NF-kappaB binding activity, whereas salicylates exert its effect by inhibiting iNOS expression at the translational or posttranslational level.

Methotrexate suppresses the interleukin-6 induced generation of reactive oxygen species in the synoviocytes of rheumatoid arthritis.

Various cytokines and reactive oxygen species (ROS) play a fundamental role in the inflammatory and immunologic processes of rheumatoid arthritis (RA). Methotrexate (MTX) is one of the disease-modifying anti-rheumatic drugs and its effect may be partly due to the modulation of immunologic or inflammatory reactions by some cytokines. In the present study, we investigated the effects of MTX on the gene expression and synthesis of interleukin-6 (IL-6), and the proliferative activity and the production of ROS in the fibroblast-like synoviocytes (FLSs) obtained from the patient of RA. The expression or production of IL-6 was induced spontaneously, and augmented by the addition of recombinant human IL-6 or recombinant human IL-1 beta and TNF-alpha in FLSs. These spontaneous and augmented IL-6 expressions or productions were suppressed by treatment with low-concentration of MTX (1 microg/ml). Also, IL-6 stimulated the proliferation of FLSs, and this IL-6 driven proliferation was inhibited with the treatment of MTX or N-acetylcysteine (NAC, 1 mM). Furthermore, ROS production in FLSs was increased significantly by IL-6, and its effect was also abrogated in the presence of MTX or NAC. These results suggest that inflammatory reaction in the synovium of RA patients could be augmented by the autocrine or other cytokine-induced production of IL-6 with subsequent generation of ROS in the synoviocytes, and the modulations of IL-6 synthesis and ROS production may contribute to the therapeutic effects of MTX for RA.

Serologic responses of Korean soldiers serving in malaria-endemic areas during a recent outbreak of Plasmodium vivax.

Anti-Pv200 antibody levels were assessed in samples from endemic areas of Plasmodium vivax malaria in the Republic of Korea (ROK), using an indirect enzyme-linked immunosorbent assay (ELISA) method. Asymptomatic carriers of P. vivax were detected using nested polymerase chain reaction (PCR) of blood samples. Anti-Pv200 antibody levels in 20 vivax malaria patients (optical density +/- standard deviation [OD +/- SD] values 1.85 +/- 0.29 of IgG isotype and 1.33 +/- 1.33 of IgM isotype) were markedly higher than those of uninfected, malaria-naive controls (0.08 +/- 0.16 of IgG isotype and 0.04 +/- 0.04 of IgM isotype). Antibody levels for 7 out of 8 soldiers with a recent malaria infection were sustained above the cut-off values for 4 months after successful treatment. Analysis of serum collected from 40 healthy, asymptomatic soldiers who had a P. vivax malaria attack within 3 months after our sampling, revealed 11 antibody-positive samples (27.5%), compared to 5 positive samples (12.5%) collected from a random selection of 40 soldiers. Among a larger pool of 1,713 soldiers who had served in high-risk areas for P. vivax transmission, 15% were antibody positive. Among 1,000 blood samples from asymptomatic soldiers who had served in the high-risk areas, 4 samples (0.4%) were parasite positive, as determined by nested PCR. Our results show that anti-Pv200 antibody levels can provide useful information in the late diagnosis of P. vivax malaria infection in a previously naive population and also in large seroepidemiologic studies. Furthermore, our results suggest that asymptomatic P. vivax carriers could be important in the current outbreak of malaria in Korea.

Hepatic ischemia/reperfusion in rats induces iNOS gene transcription by activation of NF-kappaB.

It has been known that many immediately early genes are expressed during ischemia/reperfusion (I/R) injury. Here, employing a model of hepatic I/R, we show that inducible nitric oxide synthase (iNOS) is induced via the activation of nuclear factor kappaB (NF-kappaB) after I/R in rat liver. When liver was subjected to ischemia followed by reperfusion, but not ischemia alone, an NF-kappaB complex composed of p50/p65 heterodimer and p50 homodimer was rapidly activated within 1 h and remained elevated for up to 3 h, and then tended to decline after 5 h of reperfusion. Also, the expression of iNOS mRNA was initiated after 1 h and continued to increase after 5 h of reperfusion during the time course studied. This upregulated iNOS mRNA expression coincides with increased iNOS enzyme activity and NF-kappaB binding activity after hepatic I/R. Administration of N-acetylcysteine (NAC, 20 mg/kg i.v. 10 min before reperfusion), an antioxidant, not only significantly inhibited the expression of iNOS mRNA but also blocked upregulated NF-kappaB binding activity after reperfused liver. These results suggest that NF-kappaB is activated by oxidative stress during hepatic I/R and may play a significant role in the induction of the iNOS gene.

Aldosterone directly induces Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of rat.

The change of blood pressure and the induction of Na, K-ATPase alpha 1-subunit mRNA have been investigated in the renal cortex of aldosterone-treated hypertensive rat. The increase of blood pressure by aldosterone-treatment for 25 days was decreased by the treatment of amiloride or spironolactone. The level of Na, K-ATPase alpha 1-subunit mRNA of the renal cortex in aldosterone-treated rat was increased than that in the control, and its increase was repressed by treatment of spironolactone, but not altered by the treatment of amiloride. This result suggests that the increase of Na, K-ATPase alpha 1-subunit mRNA in the renal cortex of aldosterone-treated hypertensive rat may be related with the direct induction of Na, K-ATPase mRNA without the increase of Na-traffic through Na-channel.

Regulation of Na,K-ATPase activity in renal basolateral membrane of 1-clip-1-kidney hypertensive rat.

The changes of Na,K-ATPase activity and its regulation have been investigated in the renal cortex of 1-clip-1-kidney hypertensive rat. Ouabain-sensitive Na,K-ATPase activity (Emax) and [3H]ouabain-binding site (Bmax) in the hypertensive rat were slightly increased than those in the control. The levels of Na,K-ATPase alpha 1- and beta 1-subunit mRNA of the renal cortex in hypertensive rat were more increased than those in the control. Their increases were repressed by actinomycin-D, but not altered or more increased by cycloheximide. These results suggest that the increase of Na,K-ATPase activities and ouabain binding sites in 1-clip-1-kidney hypertensive rat may be correlated with the increases of gene expression in transcription level and/or of mRNA stability of Na,K-ATPase.

Aldosterone stimulates Na,K-ATPase activity in basolateral membrane of rat kidney.

The changes of Na,K-ATPase activity and its regulation have been investigated in the renal cortex and its basolateral membrane of aldosterone-induced hypertensive rat. Ouabain-sensitive Na,K-ATPase activity and [3H]ouabain-binding site (Bmax) in the hypertensive rat were significantly increased than those in the control. The levels of Na,K-ATPase alpha 1- and beta 1-subunit mRNA of the renal cortex in hypertensive rat were more increased than those in the control, and their increases were repressed by actinomycin-D. These results suggest that the increase of Na,K-ATPase activities and ouabain binding sites in aldosterone-induced hypertensive rat may be correlated with transcriptional regulation of Na,K-ATPase gene expression.

Novel pre-C/C gene mutants of hepatitis B virus in chronic active hepatitis: naturally occurring escape mutants.

We have analysed serum samples taken from hepatitis B virus (HBV) e antigen (HBeAg)-positive and HBeAg-negative chronic active hepatitis (CAH) patients by PCR using primers spanning the pre-core/core (C) and pre-S1/S2 ORFs. Nucleotide sequence analysis showed that among 18 HBV-infected CAH patients, 11 had virus with a G to A mutation (nucleotide 1896; leading to the formation of a stop codon) and one patient also had virus with an additional G to A mutation three nucleotides downstream (nucleotide 1899). HBV from three patients that were HBeAg-negative showed a 1 bp deletion at nucleotide 1937, causing pre-termination of the C gene. Mutation frequencies in the sequences identified as coding for cytotoxic T lymphocyte epitopes, B cell epitopes, CD4+ helper T cell epitopes and arginine-rich regions of the HBV C peptide were investigated. Mutations were more frequently identified in these regions, suggesting that the mutations might have been selected as a result of immune responses.

Gradual accumulation of mutations in precore core region of HBV in patients with chronic active hepatitis: implications of clustering changes in a small region of the HBV core region.

The sequence in the precore and core region of the hepatitis B virus (HBV) genome in the serum of five chronic active hepatitis patients at four different stages in each individual were studied by polymerase chain reaction and DNA sequencing to determine the prevalence and type of precore and core mutants in each chronic active hepatitis (CAH) patient. Gradual changes of the virus genome in each CAH patient in precore and core regions were identified. Except for the virus from one patient, the mutant viruses showed gradual changes of genome sequences, which resulted in the generation of stop codons at the precore and core region, causing the association of active hepatitis in each patient even in the presence of anti-HBe. Mutational hot spots in the core region, which includes a clustering of changes in a small region of 14 amino acids (codons 84-97 from the start of the core gene) were found in all patients. This region of mutational hot spots in the core might be a major target of cytotoxic T lymphocytes (CTL), which has evolved under the pressure of immune selections, and these mutants might play a important role in the pathogenesis of viral hepatitis.