Lactational exposure to perfluorooctane sulfonate remains a potential risk in brain function of middle-aged male mice.

Perfluorooctane sulfonate (PFOS) exerts adverse effects on neuronal development in young population. Limited evidences have shown that early-life PFOS exposure holds a potential risk for developing age-related neurodegenerative diseases such as Alzheimer's disease later in life. The present study investigated the effects of lactational PFOS exposure on cognitive function using one-year-old mice. Dams were exposed to PFOS (1 mg/kg body weight) through lactation by gavage. Male offspring were used for the behavior test battery to assess cognitive function. Western blot analysis was conducted to measure the levels of proteins related to the pathogenesis of Alzheimer's disease. PFOS-exposed mice displayed a mild deficiency in social recognition. In the hippocampus, the expression of tau protein was significantly increased. These results underline a mild effect of developing PFOS exposure on cognitive function and neurodegeneration. The present study presents the long-lasting effects of PFOS in middle-aged period and warrants a potential aftermath.

Mesenchymal-epithelial transition Exon 14-Skipping Mutation-positive Invasive Mucinous Adenocarcinoma of the Lung: First Case Treated with Mesenchymal-epithelial Transition-tyrosine Kinase Inhibitors.

Mesenchymal-epithelial transition (MET) exon 14-skipping mutation (METex14) is rare in pulmonary invasive mucinous adenocarcinomas (IMAs), and the clinical impact of MET-tyrosine kinase inhibitors (TKIs) remains unknown. We herein report a 75-year-old woman with IMA harboring METex14 who was treated with the MET-TKI tepotinib. The lung tumor regressed over six months; however, the patient ultimately died of exacerbated interstitial lung disease (ILD), possibly associated with tepotinib. An autopsy revealed diffuse alveolar damage in preexisting chronic fibrosis. We discuss how to pre-evaluate ILD deterioration risks and monitor TKI-induced lung toxicity during treatment.

The role of autonomously secreted PGE2 and its autocrine/paracrine effect on bone matrix mineralization at the different stages of differentiating MC3T3-E1 cells.

Bone is consisted of osteoblast-linage cells, bone-forming cells in various differentiation stages. However, it is not fully understood how communicate and interact these cells immigrated from bone marrow. In this study, we showed that prostaglandin E2 (PGE2) had a role in autonomous modification of matrix mineralization in osteoblastic cell line, MC3T3-E1, and interactions across the cells in different differentiation stages. Analysis using LC-MS/MS and inhibitors showed the autonomous secretion of PGE2 among the prostanoids in differentiation stages and that depend on COX-2, a key enzyme for production of PGE2. Treatment with inhibitors of PGE2 receptors and COX-2 indicated that secreted PGE2 regulates matrix mineralization in an autocrine/paracrine manner. In addition, we showed that the expression profile of PGE2 receptors (EP1-EP4) and PGE2 effects on matrix mineralization derived from it changed during cell differentiation. Treatment with inhibitors of PGE2 signaling in the early differentiation stage of MC3T3-E1 cells induced significant changes in matrix mineralization several days after. Stimulation with the extracts from culture medium of the matured cells including PGE2 and co-culture with the matured cells secreting PGE2 significantly promoted matrix mineralization of the early stage cells, in contrast, treatment with inhibitor of COX-2 and PGE2 receptors failed to do so. These results support that PGE2 plays important roles in the interaction system of osteoblast-linage cells in bone tissue to regulate matrix mineralization reflecting condition of bone-forming cells, that is, population and maturation.

Regulation of MC3T3-E1 differentiation by actin cytoskeleton through lipid mediators reflecting the cell differentiation stage.

Actin cytoskeleton is reported to be related in various functions of osteoblast, bone-forming cell. However the function of actin cytoskeleton in osteoblasts is not fully understood, since bone formation is derived from extracellular interactions of functional proteins produced from osteoblasts, including osteocalcin (Ocn), and it is a result of closely and complex organized sequence of biochemical events. In this study, we showed that actin cytoskeleton of MC3T3-E1 cells functioned in recognition of cell condition and regulation of extracellular matrix mineralization, bone formation. Maturation of MC3T3-E1 cells by 14 days of culture reduced F-actin filaments, while induced expression of Ocn mRNA known as late stage differentiation marker and matrix mineralization, terminal stage of cell differentiation. The disruption of actin cytoskeleton with Cyto D in immature MC3T3-E1 cells significantly increased expression of Ocn mRNA in 24 h. Both PTX-induced inhibition of signal transduction through GPCRs and celecoxib-induced suppression of lipid mediators in immature MC3T3-E1 cells reduced actin filaments and suppressed matrix mineralization. Furthermore, addition of lipid mediators extracted from culture mediums of differentiated MC3T3-E1 cells by Bligh-Dyer method induced actin cytoskeleton reorganization and matrix mineralization change in MC3T3-E1 cells. Taken together, our data suggest that actin cytoskeleton of MC3T3-E1 cells regulates activation of developmental pathway reflecting cell differentiation stages through lipid mediators. The function we identified is important for bone formation tightly regulated by mechanical stress, since actin cytoskeleton is also known as a mechanosensor of osteoblasts.

BinCARD2 as a positive regulator of interferon response in innate immunity.

Innate immunity is a system that recognizes primarily and excludes pathogenic microorganism. MAVS/IPS-1/Cardif/Visa functions as an adapter protein for RIG-I like receptors (RLRs) and plays a key role in the production of antiviral proteins, interferons (IFNs), for RNA viruses. However, the activation mechanism is not fully understood. Here, we show that BinCARD isoform2 (BinCARD2), carrying CARD domain structure like MAVS, functions in innate immune response. Knockdown of BinCARD2 reduced the RLR ligand-induced expression of IFN-ß mRNA and activation of the IFNB promoter. The activation of the IFNB promoter by overexpression of MAVS or TBK1 was suppressed by silencing of BinCARD2, but no effect on IFNB promoter activation by overexpression of TRIF or constitutive activated IRF-3. Furthermore, we confirmed that BinCARD2 protein associated with MAVS but not TBK1 by immunoprecipitation and colocalized with MAVS. Accordingly, we investigated whether BinCARD2 was involved in MAVS activation and showed that siBinCARD2 did not affect RIG-I/MAVS binding but impaired the MAVS oligomerization. Moreover, we infected A549 cells with vesicular stomatitis virus (VSV) and found that induction of IFN-ß and IL-6 mRNA after VSV infection was decreased by BinCARD2 knockdown. Thus, these data may suggest that BinCARD2 associates with MAVS to positively modulate the oligomerization in the RIG-I like receptors pathway and activates innate immune response.

Evaluation of 4ß-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice.

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4ß-hydroxycholesterol (4ß-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4ß-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4ß-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4ß-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4ß-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4ß-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4ß-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4ß-HC is a more reliable biomarker of hepatic CYP3A activity.

Osteoblast differentiation is functionally associated with decreased AMP kinase activity.

Osteoblasts, originating from mesenchymal stem cells, play a pivotal role in bone formation and mineralization. Several transcription factors including runt-related transcription factor 2 (Runx2) have been reported to be essential for osteoblast differentiation, whereas the cytoplasmic signal transduction pathways controlling the differentiation process have not been fully elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis, polarity, and division. Recent lines of evidence have indicated that the activity of the catalytic alpha subunit of AMPK is regulated through its phosphorylation by upstream AMPK kinases (AMPKKs) including LKB1. Here, we explored the role of AMPK in osteoblast differentiation using in vitro culture models. Phosphorylation of AMPKalpha was significantly decreased during osteoblastic differentiation in both primary osteoblasts and MC3T3-E1, a mouse osteoblastic cell line. Conversely, the terminal differentiation of primary osteoblasts and MC3T3-E1 cells, represented by matrix mineralization, was significantly inhibited by glucose restriction and stimulation with metformin, both of which are known activators of AMPK. Matrix mineralization of MC3T3-E1 cells was also inhibited by the forced expression of a constitutively active form of AMPKalpha. Metformin significantly inhibited gene expression of Runx2 along with osteoblast differentiation markers including osteocalcin (Ocn), bone sialo protein (Bsp), and osteopontin (Opn). Thus, our present data indicate that differentiation of osteoblasts is functionally associated with decreased AMPK activity.