In human T cells mifepristone antagonizes glucocorticoid non-genomic rapid responses in terms of Na(+)/H(+)-exchange 1 activity, but not ezrin/radixin/moesin phosphorylation.

Glucocorticoids (GCs) and progesterone have been employed as immunosuppressive agents during pregnancy for many years. Intracellular acidification by GCs is due to a rapid non-genomic inhibition of membrane Na(+)/H(+)-exchange 1 (NHE1) activity and is followed by immunosuppression of PHA-stimulated proliferation. NHE1 is tethered to the cortical actin cytoskeleton through ezrin/radixin/moesin (ERM) proteins within lipid rafts; these regulate cell shape, migration and resistance to apoptosis. We explored whether mifepristone (RU486), an antagonist of GCs in T cells, is able to completely block rapid non-genomic responses, namely NHE1 activity and the phosphorylation C-terminal residues of ERM proteins at threonine (cp-ERM). GCs stimulate a rapid non-genomic cp-ERM response in cells within 5min. RU486 antagonized the GC-induced rapid decrease in NHE1 activity, and arrested PHA-stimulated T cells at G0/G1 phase but had no effect on the rapid increase in cp-ERM, which persisted for 24h. However, the cp-ERM response was blocked by staurosporine in both resting and GC stimulated cells. The results of RU486 antagonized the GC induced rapid decrease in NHE1 ion transport activity, but not the increase cp-ERM. This suggests that RU486 in T cells exerts its antagonistic effects at NHE1 containing plasma membrane sites and not where cp-ERM links lipid rafts to cortical cytoskeletons.

Higher susceptibility to aflatoxin B(1)-related hepatocellular carcinoma in glycine N-methyltransferase knockout mice.

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N-methyltransferase (GNMT) interacts with aflatoxin B(1) (AFB(1)) and reduces both AFB(1)-DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt-/-) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt-/- mice are susceptible to AFB(1) carcinogenesis, and that the absence of Gnmt expression may accelerate AFB(1)-induced liver tumorigenesis. We inoculated Gnmt-/- and wild-type mice intraperitoneally with AFB(1) at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt-/- mice were diagnosed as having HCC, ∼6 months earlier than AFB(1)-treated wild-type mice. Results from microarray and real-time PCR analyses indicate that five detoxification pathway-related genes were downregulated in AFB(1)-treated Gnmt-/- mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB(1)-related HCC in Gnmt-/- mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.

Deficiency of glycine N-methyltransferase results in deterioration of cellular defense to stress in mouse liver.

PURPOSE: Previously, we reported that glycine N-methyltransferase (GNMT) interacts with benzo[a]pyrene (BaP) and inhibits BaP-DNA adducts formation. In addition, Gnmt knockout (Gnmt(-/-)) mice developed chronic hepatitis and hepatocellular carcinoma (HCC). The aims of this study were to understand the gene expression profile of Gnmt(-/-) mice and to study the interaction between BaP and GNMT deficiency in vivo. EXPERIMENTAL DESIGN: Gene expression profiles of Gnmt(-/-) mice were analyzed by 2-D PAGE and real-time PCR. Both wild-type and Gnmt(-/-) mice were challenged with BaP and sacrificed at the age of 13 months. RESULTS: Compared with the wild-type mice, proteins involved in the anti-oxidation/detoxification response, glycolytic energy metabolism and one-carbon metabolism pathways were down-regulated significantly in Gnmt(-/-) mice. Malondialdehyde assay showed that lipid peroxidation was significantly increased in the Gnmt(-/-) mice liver. H(2)O(2) treatment demonstrated that the survival rate of HuH-7 cells overexpressing GNMT was significantly higher than the controls. BaP challenge experiments showed that 71.4% (5/7) of male and all (7/7) female Gnmt(-/-) mice developed HCC, while only 16.7% (1/6) of male and 20% (1/5) of female wild-type mice had HCC. CONCLUSION AND CLINICAL RELEVANCE: GNMT regulates genes related to detoxification and anti-oxidation pathways. BaP is a liver cancer carcinogen especially during GNMT deficiency.

Non-genomic rapid inhibition of Na+/H+-exchange 1 and apoptotic immunosuppression in human T cells by glucocorticoids.

Glucocorticoids (GCs) have been employed as immunosuppressive agents for many years. However, it is still unclear how GCs instantly uncouple T cells from acute stressful inflammatory. In terms of time scale, the genomic activity of the classic GC receptor cannot fulfill this role under crisis; but a rapid non-genomic response can. In a previous study, intracellular acidification was found to be due to a rapid non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) and this event led to the immunosuppression of T cell proliferation by progesterone. The aim of this study was to examine whether there is a rapid acidification response caused by an inhibition of NHE1 activity and to explore the differential non-genomic effect on immunosuppression of hydrocortisone and dexamethasone. The IC(50) values for NHE1-dependent pH(i) recovery by hydrocortisone and dexamethasone are 250 and 1 nM, respectively. Co-stimulation of GCs with phytohemagglutinin (PHA) is able to inhibit PHA-induced IL-2 secretion, IL-4 secretion, and T-cell proliferation. Furthermore, apoptosis in PHA-activated T cells is not induced by hydrocortisone but by dexamethasone. The mechanism of immunosuppression on proliferation by dexamethasone was found to be different of hydrocortisone and seems to involve cytotoxicity against T cells. Moreover, apoptosis induced by dexamethasone and impermeable dexamethasone-bovine serum albumin suggests that the apoptotic immunosuppression occurs through both the plasma membrane and cytoplasmic sites. The rapid inhibitory responses triggered by GCs would seem to release T cells instantly when an acute stress-related response is needed. Nonetheless, the apoptotic immunosuppression by dexamethasone is attributable to its severe cytotoxicity.

Inducible nitric oxide synthase expression and plasma bilirubin changes in rats under intermittent hypoxia treatment.

It has been reported that intermittent hypoxia treatment prevents oxidative injuries to the brain and protects the heart against ischemia-reperfusion injury. Both anti-oxidative defensive systems and prevention of free intracellular calcium overload might be the result of intermittent hypoxia. Thus, the purpose of this study was to explore the effects of intermittent hypoxia (8 h at 12 % O2 per day) for 0, 7 or 14 days on inducible nitric oxide synthase (iNOS) expression in the spleen and on splenic calcium response to the mitogen phytohemagglutinin (PHA). The results demonstrated that administration of intermittent hypoxia for 7 days caused severe hemolysis of erythrocytes in the spleen and the hemolytic condition was ameliorated by intermittent hypoxia for 14 days. However, a significant decline in splenic weight and an increase in plasma total bilirubin levels appeared in rats after hypoxia for 14 days. No calcium response to PHA was observed in splenocytes obtained from rats after intermittent hypoxia for 7 days. After intermittent hypoxia for 14 days, the calcium response to PHA was restored to the level of the controls. Intermittent hypoxia for 7 days was able to induce higher iNOS expression in splenic tissues than hypoxia for 14 days. These results suggested that intermittent hypoxia for 14 days appeared to involve acclimatization that protects the rats from oxidative injury through less hemolysis and iNOS expression in splenic tissues and by the presence of more bilirubin in the plasma. The increase in plasma total bilirubin levels might be the cause of induced adaptation to chronic intermittent hypoxia.

Activation and up-regulation of phospholipase D expression by lipopolysaccharide in human peripheral T cells.

In a previous study, we showed that bacterial LPS activates protein kinase C (PKC) and causes an intracellular pH (pHi) increase, but does not elevate intracellular calcium ([Ca2+]i) in human peripherial T cells. Hence this study aimed to investigate whether the activation of PKC was resulted from phospholipase D (PLD) catalysis by LPS. The activity of PLD was measured by the production of 3H-phosphatidylethanol from phosphatidic acid (PA), and the expression of PLD or IL-2 Ralpha was determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Enzyme-linked immunosorbent assay (ELISA) was used to analyze IL-2 and IL-4. Phytohemagglutinin (PHA), and phorbol 12-myristate 13-acetate (PMA) were used as controls. Our results indicated that (1) LPS-stimulated pHi elevation was PKC dependent; (2) After 30 min stimulation, LPS increased PLD activity via a measured production of 3H-phosphatidylethanol from phosphatidic acid and the initiation of PLD1a mRNA expression started; (2) LPS stimulated IL-2 R expression but not IL-2 and IL-4 secretion. Our findings suggested that the stimulation of PLD activity and its mRNA expression by LPS might be required for IL-2 R expression and a sustained PKC dependent pHi elevation but not for the secretion of IL-2 or IL-4 in human T cells. This indicated that LPS might enhance T cell adaptive immunity to resist Gram-negative bacterial infection.