MKRN2 is a novel ubiquitin E3 ligase for the p65 subunit of NF-κB and negatively regulates inflammatory responses.

Activation of NF-κB transcription factor is strictly regulated to prevent excessive inflammatory responses leading to immunopathology. However, it still remains unclear how NF-κB activation is negatively controlled. The PDZ-LIM domain-containing protein PDLIM2 is a nuclear ubiquitin E3 ligase targeting the p65 subunit of NF-κB for degradation, thus terminating NF-κB-mediated inflammation. Using yeast two-hybrid screening, we sought to isolate PDLIM2-interacting proteins that are critical for suppressing NF-κB signaling. Here we identified MKRN2, a RING finger domain-containing protein that belongs to the makorin ring finger protein gene family, as a novel p65 ubiquitin E3 ligase. MKRN2 bound to p65 and promoted the polyubiquitination and proteasome-dependent degradation of p65 through the MKRN2 RING finger domain, thereby suppressing p65-mediated NF-κB transactivation. Notably, MKRN2 and PDLIM2 synergistically promote polyubiquitination and degradation of p65. Consistently, MKRN2 knockdown in dendritic cells resulted in larger amounts of nuclear p65 and augmented production of proinflammatory cytokines in responses to innate stimuli. These results delineate a novel role of MKRN2 in negatively regulating NF-κB-mediated inflammatory responses, cooperatively with PDLIM2.

Changes in the methylation status of DAT, SERT, and MeCP2 gene promoters in the blood cell in families exposed to alcohol during the periconceptional period.

BACKGROUND: Alcohol exposure has been shown to cause devastating effects on neurobehavioral development in numerous animal and human studies. The alteration of DNA methylation levels in gene-specific promoter regions has been investigated in some studies of human alcoholics. This study was aimed to investigate whether social alcohol consumption during periconceptional period is associated with epigenetic alteration and its generational transmission in the blood cells. METHODS: We investigated patterns of alcohol intake in a prospective cohort of 355 pairs of pregnant women and their spouses who reported alcohol intake during the periconceptional period. A subpopulation of 164 families was established for the epigenetic study based on the availability of peripheral blood and cord blood DNA. The relative methylation changes of dopamine transporter (DAT), serotonin transporter (SERT), and methyl CpG binding protein 2 (MeCP2) gene promoters were analyzed using methylation-specific endonuclease digestion followed by quantitative real-time polymerase chain reaction. RESULTS: The relative methylation level of the DAT gene promoter was decreased in the group of mothers reporting above moderate drinking (p = 0.029) and binge drinking (p = 0.037) during pregnancy. The relative methylation level of the DAT promoter was decreased in the group of fathers reporting heavy binge drinking (p = 0.003). The relative methylation levels of the SERT gene promoter were decreased in the group of newborns of light drinking mothers before pregnancy (p = 0.012) and during pregnancy (p = 0.003). The methylation level in the MeCP2 promoter region of babies whose mothers reported above moderate drinking during pregnancy was increased (p = 0.02). In addition, methylation pattern in the DAT promoter region of babies whose fathers reported heavy binge drinking was decreased (p = 0.049). CONCLUSIONS: These findings suggest that periconceptional alcohol intake may cause epigenetic changes in specific locus of parental and newborn genomes as follows: Alcohol consumption decreases the methylation level of the DAT promoter region of the parent themselves, maternal alcohol drinking during the periconceptional period decreases the methylation level of the SERT promoter region of newborns, and maternal alcohol consumption increases the methylation level of the MeCP2 promoter region of newborns.

Characterization of phosphatidylethanol blood concentrations for screening alcohol consumption in early pregnancy.

OBJECTIVE: Phosphatidylethanol (PEth) is formed endogenously by the direct action of ethanol, and has a half-life long enough to make it a reliable biomarker of alcohol exposure in early pregnancy. In this study, we aimed to characterize PEth blood concentrations to differentiate different levels of alcohol exposure in pregnant women. METHODS: The study consisted of 305 consecutive pregnant women who had been referred to our hospital for antenatal care. Of them, 117 self-reported alcohol ingestion in the first trimester of pregnancy and 188 were abstainers. Total PEth concentration in whole blood was quantified by liquid chromatography-mass spectrometry (LC-MS/MS). Alcohol ingestion was classified according to the United States National Institute on Alcohol Abuse and Alcoholism into light drinkers: ≤ 3 drinks/week, moderate drinkers: 3-7 drinks/week, and heavier drinkers: > 7 drinks/week (a standard drink = 14 g of ethanol). RESULTS: Participants had quantifiable PEth blood levels 3-4 weeks after the last drink. There were 4.8% abstainers who had positive PEth concentrations; all of them reported a positive history of alcohol consumption before conception. PEth blood concentrations were significantly correlated to drinks per occasion (r = 0.44; P < 0.001) and days drinking per week (r = 0.34; P < 0.001). However, almost 74% of participants with ≤ 3 drinks/week of alcohol, and 46% with 3-7 drinks/week, had PEth blood concentrations below the lower limit of quantification (LLOQ). The area under the curve (AUC) generated by a receiver operation characteristic curve (ROC) analysis increased as the cutoff value of PEth blood concentration increased. However, the cutoff values were below or close to the LLOQ. CONCLUSIONS: Our study presents a formal characterization of PEth blood concentrations for screening alcohol ingestion in first-trimester pregnant women. However, caution is recommended for overrepresenting either negative or positive results.

Blood levels of phosphatidylethanol in pregnant women reporting positive alcohol ingestion, measured by an improved LC-MS/MS analytical method.

OBJECTIVE: A reliable biomarker of low alcohol exposure during pregnancy is needed to clarify the controversy on the teratogenicity of low-to-moderate alcohol levels. METHODS: Blood samples were obtained from 13 pregnant women who self-reported alcohol ingestion between 2.5 and 20 drinks/week, and from 26 controls. Total lipids were extracted, and phosphatidylethanol (PEth) species 16:0/16:0, 16:0/18:1, and 16:0/18:1 were separated by high-performance liquid chromatography (HPLC) on a reverse-phase phenyl column. These PEth species were quantified by MS/MS using phosphatidylpropanol as internal standard, with electrospray ionization and MRM. RESULTS: PEth species were not detected in women who abstained from alcohol ingestion during pregnancy, whereas PEth-16:0/18:1 was > 5 nmol/L in those with positive alcohol ingestion. PEth species were detected for up to 4 weeks after cessation of exposure. CONCLUSIONS: PEth-16:0/18:1 was detected in pregnant women at 4-6 weeks after their last low-to-moderate alcohol ingestion, and therefore appears to be a reliable biomarker of prenatal alcohol exposure to study the teratogenicity of alcohol at these exposure levels.

Mammalian MST2 kinase and human Salvador activate and reduce estrogen receptor alpha in the absence of ligand.

Mammalian MST2 kinase plays an important role in cell proliferation, survival, and apoptosis. In search of interacting proteins of MST2, we found that estrogen receptor α (ERα) co-immunoprecipitates with MST2 and its adaptor protein human Salvador (hSAV). Using reporter assays, we observed that overexpression of MST2 and hSAV leads to ligand-independent activation of ERα in human breast cancer MCF-7 cells, which was attenuated by the knockdown of hSAV. Furthermore, using truncated mutants of hSAV, we observed that the C terminus of hSAV is necessary and sufficient for the induction of ERα transactivation. The expression of hSAV and MST2 results in the phosphorylation of ERα at serine residues 118 and 167 and represses ERα expression. We then investigated the incidence of MST2 and ERα expression with other tumor biomarkers using commercially available tissue microarrays. Among 40 breast cancer samples analyzed, 60% (24 out of 40) expressed MST2. Nineteen among the 40 cases were MST2-positive and ERα-negative, implying a correlation between expressions of MST2 with loss of ERα in breast tumor samples. This study suggests that MST and hSAV act as novel co-regulators of ERα and may play an important role in breast cancer pathogenesis.

Anti-inflammatory mechanism of ginsenoside Rh1 in lipopolysaccharide-stimulated microglia: critical role of the protein kinase A pathway and hemeoxygenase-1 expression.

Microglia activation plays a pivotal role in neurodegenerative diseases, and thus controlling microglial activation has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we showed that ginsenoside Rh1 inhibited inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated microglia, while Rh1 increased anti-inflammatory IL-10 and hemeoxygenase-1 (HO-1) expression. Suppression of microglial activation by Rh1 was also observed in the mouse brain following treatment with LPS. Subsequent mechanistic studies revealed that Rh1 inhibited LPS-induced MAPK phosphorylation and nuclear factor-κB (NF-κB)-mediated transcription without affecting NF-κB DNA binding. As the increase of pCREB (cAMP responsive element-binding protein) is known to result in suppression of NF-κB-mediated transcription, we examined whether Rh1 increased pCREB levels. As expected, Rh1 increased pCREB, which was shown to be related to the anti-inflammatory effect of Rh1 because pre-treatment with protein kinase A inhibitors attenuated the Rh1-mediated inhibition of nitric oxide production and the up-regulation of IL-10 and HO-1. Furthermore, treatment of HO-1 shRNA attenuated Rh1-mediated inhibition of nitric oxide and reactive oxygen species production. Through this study, we have demonstrated that protein kinase A and its downstream effector, HO-1, play a critical role in the anti-inflammatory mechanism of Rh1 by modulating pro- and anti-inflammatory molecules in activated microglia.