Adenine attenuates lipopolysaccharide-induced inflammatory reactions

A nucleobase adenine is a fundamental component of nucleic acids and adenine nucleotides. Various biological roles of adenine have been discovered. It is not produced from degradation of adenine nucleotides in mammals but produced mainly during polyamine synthesis by dividing cells. Anti-inflammatory roles of adenine have been supported in IgE-mediated allergic reactions, immunological functions of lymphocytes and dextran sodium sulfate-induced colitis. However adenine effects on Toll-like receptor 4 (TLR4)-mediated inflammation by lipopolysaccharide (LPS), a cell wall component of Gram negative bacteria, is not examined. Here we investigated anti-inflammatory roles of adenine in LPS-stimulated immune cells, including a macrophage cell line RAW264.7 and bone marrow derived mast cells (BMMCs) and peritoneal cells in mice. In RAW264.7 cells stimulated with LPS, adenine inhibited production of pro-inflammatory cytokines TNF-α and IL-6 and inflammatory lipid mediators, prostaglandin E₂ and leukotriene B₄. Adenine impeded signaling pathways eliciting production of these inflammatory mediators. It suppressed IκB phosphorylation, nuclear translocation of nuclear factor κB (NF-κB), phosphorylation of Akt and mitogen activated protein kinases (MAPKs) JNK and ERK. Although adenine raised cellular AMP which could activate AMP-dependent protein kinase (AMPK), the enzyme activity was not enhanced. In BMMCs, adenine inhibited the LPS-induced production of TNF-α, IL-6 and IL-13 and also hindered phosphorylation of NF-κB and Akt. In peritoneal cavity, adenine suppressed the LPS-induced production of TNF-α and IL-6 by peritoneal cells in mice. These results show that adenine attenuates the LPS-induced inflammatory reactions.

Tryptophan Negatively Regulates IgE-mediated Mast Cell Activation / 체질인류학회지

Mast cells are major immune cells in allergy to secrete allergic mediators by a degranulation process and make and secrete inflammatory lipids and cytokines in response to antigen stimulation. An amino acid tryptophan regulates immune functions. Tryptophan ameliorates inflammatory colitis in which mast cells are engaged. However, its effects on mast cells remain to be solved. We investigated the effect of tryptophan on IgE-mediated allergic responses in the mast cells and mice. IgE-mediated passive cutaneous anaphylaxis (PCA) in mice were examined. Also IgE-mediated mast cell activation responses such as degranulation of stored granules and secretion of inflammatory lipid LTB₄ and cytokines (TNF-α and IL-4) were measured. Intraperitoneal administration of tryptophan suppressed PCA in mice. Also, in the cellular level tryptophan inhibited IgE-mediated mast cell activation such as IgE-mediated degranulation and the production of LTB₄. Also, it inhibited production of inflammatory cytokines TNF-α and IL-4. In summary, tryptophan suppressed IgE-mediated allergic activation in vivo and in vitro. Tryptophan supplementation is beneficial for IgE-mediated allergy.

Dexamethasone Induces FcgammaRIIb Expression in RBL-2H3 Cells

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress FcepsilonRI-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE (FcepsilonR) I and increased the mRNA levels of the inhibitory Fc receptor for IgG FcgammaRIIb. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG (FcgammaR) I and FcgammaRIII. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced FcgammaRI and FcgammaRIII mRNA levels potently, while FcepsilonRI and FcgammaRIIb were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only FcgammaRIIb protein expression was significantly enhanced by Dex treatment, while FcgammaRI, FcgammaRIII and FcepsilonRI expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor FcgammaRIIb.

Transcriptional Regulation of the Estrogen Receptor alpha Gene by Testosterone in Cultures of Primary Rat Sertoli Cells / 대한내분비학회지

BACKGROUND: We wanted to identify the presence of the estrogen receptor (ER) alpha in Sertoli cells and gain insight on the regulation of the ER alpha gene expression by testosterone in Sertoli cells. The transcriptional regulation of the ER alpha gene was investigated in primary Sertoli cell cultures by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Primary Sertoli cell culture was performed. The expression levels of ER alpha and ER beta mRNA in Sertoli cells were detected by Northern blot, RT-PCR, immunocytochemistry and in situ hybridization. RESULTS: The ovary, testis and epididymis showed a moderate to high expression of ER alpha while the prostate, ovary and LNCap cells showed the ER beta expression. ER alpha mRNA and protein were detected in the germ cells and Sertoli cells by in situ hybridization and immunocytochemistry. The level of ER alpha mRNA was gradually decreased in a time-dependent manner after testosterone treatment, and the changes of ER alpha mRNA were dependent on the concentration of testosterone. Androgen binding protein and testosterone-repressive prostate message-2 (TRPM-2) mRNA were reduced at 24 hour by estradiol, while the transferrin mRNA was not affected. ER alpha mRNA was strongly detectable in the testes of 7 days-old-rats, but it was gradually decreased from 14 to 21 days of age. The primary Sertoli cells also showed the same pattern. The ER alpha gene expression was also regulated by testosterone in the Sertoli cells prepared from the 14- and 21-day old rats. CONCLUSIONS: These results suggest that ER alpha is transcriptionally regulated by testosterone and it may play some role in the Sertoli cells.

Anti-apoptotic role of phospholipase D isozymes in the glutamate-induced cell death

Abstract Phospholipase D (PLD) plays an important role as an effector in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. Recently, PLD2 was reported as a necessary intermediate in preventing apoptosis induced by hydrogen peroxide or hypoxia in rat pheochromocytoma (PC12) cells. The data presented here show that both PLD isozymes, PLD1 and PLD2 are also required in attenuating glutamate-induced cell death in PC12 cells. Treatment of PC12 cells with glutamate resulted in induction of apoptosis in these cells, which is accompanied by decreased PLD activity and increased ceramide concentration. Incubation of PC12 cells with exogenous C6-ceramide showed a time-dependent decrease of PLD activity. When cDNAs of PLD1 and PLD2 were transfected into PC12 cells respectively, overexpression of PLD1 or PLD2 resulted in inhibition of glutamate-induced apoptotic cell death. These data indicate that both PLD1 and PLD2 play a protective role against glutamate-induced cell death in PC12 cells.

Enhancement of ATP-induced Currents by Phospholipase D1 Overexpressed in PC12 Cells

Using phospholipase D1 (PLD1) -overexpressing PC12 (PLD1-PC12) cells, the regulatory roles of PLD1 on ATP-induced currents were investigated. In control and PLD1-PC12 cells, ATP increased PLD activity in an external Ca2+ dependent manner. PLD activity stimulated by ATP was substantially larger in PLD1-PC12 cells than in control cells. In whole-cell voltage-clamp mode, ATP induced transient inward and outward currents. The outward currents inhibited by TEA or charybdotoxin were significantly larger in PLD1-PC12 cells than in control cells. The inward currents known as Ca2+ permeable nonselective cation currents were also larger in PLD1-PC12 cells than in control cells. However, the difference between the two groups of cells disappeared in Ca2+ -free external solution, where ATP did not activate PLD. Finally, ATP-induced 45Ca uptakes were also larger in PLD1-PC12 cells than in control cells. These results suggest that PLD enhances ATP-induced Ca2+ influx via Ca2+ permeable nonselective cation channels and increases subsequent Ca2+ -activated K+ currents in PC12 cells.

Activation of epidermal growth factor receptor is responsible for pervanadate-induced phospholipase D activation

Pervanadate, a complex of vanadate and H2O2, has an insulin mimetic effect, and acts as an inhibitor of protein tyrosine phosphatase. Pervanadate-induced phospholipase D (PLD) activation is known to be dependent on the tyrosine phosphorylation of cellular proteins and protein kinase C (PKC) activation, and yet underlying molecular mechanisms are not clearly understood. Here, we investigated the signaling pathway of pervanadate-induced PLD activation in Rat2 fibroblasts. Pervanadate increased PLD activity in dose- and time- dependent manner. Protein tyrosine kinase inhibitor, genistein, blocked PLD activation. Interestingly, AG-1478, a specific inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR) blocked not only the PLD activation completely but also phosphorylation of p38 mitogen- activated protein kinase (MAPK). However, AG-1295, an inhibitor specific for the tyrosine kinase activity of pletlet drived growth factor receptor (PDGFR) did not show any effect on the PLD activation by pervanadate. We further found that pervanadate increased phosphorylation levels of p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). SB203580, a p38 MAPK inhibitor, blocked the PLD activation completely. However, the inhibitions of ERK by the treatment of PD98059 or of JNK by the overexpression of JNK interacting peptide JBD did not show any effect on pervanadate-induced PLD activation. Inhibition or down-regulation of PKC did not alter the pervanadate-induced PLD activation in Rat2 cells. Thus, these results suggest that pervanadate-induced PLD activation is coupled to the transactivation of EGFR by pervanadate resulting in the activation of p38 MAP kinase.

Specialized Functions and Hormonal Regulation of Sertoli Cell / 대한내분비학회지

No abstract available.

Association of castration-dependent early induction of c-myc expression with a cell proliferation of the ventral prostate gland in rat

The protooncogene c-myc is known to be associated with both cell proliferation and apoptosis. The possible cellular affects of castration on the ventral prostate gland of rat as well as the relationship to a castration induced c-myc expression were examined. Levels of c-myc mRNA in the ventral prostate gland peaked at 6 h (early induction) and 48 h (late induction) after castration, respectively. Castration-induced DNA fragmentation was not observed at an early induction of c-myc mRNA. DNA fragmentation appeared to be testosterone-dependent. On the other hand, cellular DNA synthesis measured by [3H]thymidine uptake in the ventral prostate gland was increased to maximum at 6 h after castration. These results suggest that an early induction of c-myc mRNA in ventral prostate gland after castration is closely associated with cell proliferation of the gland.