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Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.

SPRY domain-containing SOCS box protein 1 (SPSB1) is an E3 ligase adaptor protein with unknown functions in cancer cells. In this study, we found that SPSB1 knockdown markedly decreased the viability and migration of ovarian cancer cells, while ectopic SPSB1 overexpression in IL-3-dependent Ba/F3 cells significantly increased their proliferation rate compared with empty vector-transfected cells. SPSB1 knockdown significantly elevated p21 protein and mRNA levels and induced apoptosis in ovarian cancer cells, as evidenced by increased levels of cleaved PARP and decreased levels of Bcl-2. Notably, mechanistic investigations revealed that SPSB1 accelerated p21 destabilization by directly interacting with p21 and promoting its ubiquitin-mediated proteasomal degradation. Taken together, our findings provide novel insights into the role of SPSB1 in ovarian cancer cells.

Ischemic preconditioning (IPC) significantly reduces ischemia–reperfusion injury in the brain by inducing ischemic tolerance. Although emerging evidence suggests that microRNAs (miRNAs) contribute to the pathogenesis of brain ischemia and IPC-induced neuroprotection, the role of miRNAs and their underlying mechanisms are still unclear. IPC was induced in male C57BL/6 mice by brief bilateral common carotid artery occlusion. After 24 h, mice underwent transient middle cerebral artery occlusion followed by 3 h of reperfusion. Expression levels of messenger RNAs (mRNAs) and proteins were examined in the ipsilateral cortex, and mimics and inhibitors of selective miRNAs were transfected into Neuro-2a cells before oxygen–glucose deprivation (OGD). Post-IPC miRNA expression profiling identified neuroprotection-associated changes in miRNA expression in the ipsilateral cortex after ischemic stroke. Among them, miR-33-5p and miR-135b-5p were significantly downregulated by IPC. Inhibition of miR-33-5p and miR-135b-5p expression protected Neuro-2a cells from OGD-induced apoptosis. Inhibition of these two miRNAs significantly increased mRNA and protein levels of ATP-binding cassette subfamily A member 1 (ABCA1), and a binding assay showed that these two miRNAs showed specificity for Abca1 mRNA. Overexpression of ABCA1 decreased the Bax/Bcl2 mRNA ratio and activation of caspase-9 and caspase-3, whereas knockdown of ABCA1 expression increased the Bax/Bcl2 mRNA ratio and the percentage of Neuro-2a cells with a loss of mitochondrial membrane potential after OGD-treatment. In conclusion, ABCA1 expression is regulated by miR-33-5p and miR-135b-5p. Increased ABCA1 expression following IPC exerts a protective influence against cerebral ischemia via suppression of a mitochondria-dependent apoptosis pathway.

Ischemic preconditioning (IP) reduces brain damage after subsequent ischemic strokes by activating endogenous protective mechanisms in rodents. Transient ischemic attack (TIA) induces tolerance in the human brain after ischemic strokes; defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes. Iron transported across the blood–brain barrier (BBB) is required for brain functions, including myelination, and its levels should be finely regulated to avoid harmful effects. This study aimed to determine whether IP enhances repair processes by modulating iron metabolism during the post-stroke chronic phase. Male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke. There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of tight junction proteins of BBB, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins, transferrin receptor 1 and DMT1, were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.

BACKGROUND AND PURPOSE The pathogenesis of moyamoya disease (MMD) remains poorly understood, and no reliable molecular biomarkers for MMD have been identified to date. The present study aimed to identify epigenetic biomarkers for use in the diagnosis of MMD. METHODS We performed integrated analyses of gene expression profiles and DNA methylation profiles in endothelial colony forming cells (ECFCs) from three patients with MMD and two healthy individuals. Candidate gene mRNA expression and DNA methylation status were further validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and pyrosequencing analysis of an expanded ECFC sample set from nine patients with MMD and ten controls. We evaluated the diagnostic accuracy of the potential biomarkers identified here using receiver operating characteristic curve analyses and further measured major angiogenic factor expression levels using a tube formation assay and RT-qPCR. RESULTS Five candidate genes were selected via integrated analysis; all five were upregulated by hypomethylation of specific promoter CpG sites. After further validation in an expanded sample set, we identified a candidate biomarker gene, sortilin 1 (SORT1). DNA methylation status at a specific SORT1 promoter CpG site in ECFCs readily distinguished patients with MMD from the normal controls with high accuracy (area under the curve 0.98, sensitivity 83.33%, specificity 100%). Furthermore, SORT1 overexpression suppressed endothelial cell tube formation and modulated major angiogenic factor and matrix metalloproteinase-9 expression, implying SORT1 involvement in MMD pathogenesis. CONCLUSION s Our findings suggest that DNA methylation status at the SORT1 promoter CpG site may be a potential biomarker for MMD.

Purpose Glioblastoma multiforme (GBM) is the most common adult primary intracranial tumor. The remarkable features of GBM include central necrosis. MicroRNAs (miRNAs) have been considered as diagnostic/prognostic biomarkers for many cancers, including glioblastoma. However, the effect of necrosis on the miRNA expression profile and predicted miRNA-mRNA regulatory information remain unclear. The purpose of this study is to examine the effect of necrotic cells on the modulation of miRNA and mRNA expression profiles and miRNA-mRNA network in CRT-MG cells. Materials and Methods We used human astroglioma cells, CRT-MG, treated with necrotic CRT-MG cells to examine the effect of necrosis on the modulation of miRNA and mRNA by next-generation sequencing. For preparation of necrotic cells, CRT-MGcellswere frozen and thawed through cycle of liquid nitrogen–water bath. The putative miRNA-mRNA regulatory relationshipwas inferred through target information, using miRDB. Results The necrotic cells induced dysregulation of 106 miRNAs and 887 mRNAs. Among them, 11 miRNAs that had a negative correlation value of p < 0.05 by the hypergeometric test were screened, and their target mRNAs were analyzed by Gene Ontology enrichment analysis. Using the Kyoto Encyclopedia of Genes and Genomes database, we also found several necrotic cell treatment-activated pathways that were modulated by relevant gene targets of differentially expressed miRNAs. Conclusion Our result demonstrated that dysregulation of miRNA and mRNA expression profiles occurs when GBM cells are exposed to necrotic cells, suggesting that several miRNAs may have the potential to be used as biomarkers for predicting GBM progression and pathogenesis.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-Nacetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer.

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long‐chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very‐long acyl chain ceramides with concomitant increase of long chain bases and C16‐ceramides, were more susceptible to dextran sodium sulphate‐induced colitis, and their survival rate was markedly decreased compared with that of wild‐type littermates. Using mixed bone‐marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule‐A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco‐2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2‐knockdown via CRISPR‐Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long‐chain bases and C16‐ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC‐dextran levels, indicating that altered SLs including deficiency of very‐long‐chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming α1 subunit and, through its C-terminal region, scaffolds the β subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the α1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

Recently, sphingolipid derivatives, such as ceramide and sphingosine‑1‑phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF‑7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal‑regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF‑7 cell proliferation. This effect was partially reversed by co‑treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6‑mediated decrease in MCF‑7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF‑7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16‑ceramide, which is generated by CerS6.

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179 phosphorylation, aphidicolin decreased eNOS-Ser116 phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116 dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566 phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116 and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.

Purpose Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties. However, the precise regulatory mechanisms controlling metastasis genes and their role in metastatic transformation are largely unknown. To address epigenetically-regulated gene products involved in ovarian cancer metastasis, we examined the mechanisms regulating mucin 13 (MUC13) expression and its influence on aggressive behaviors of ovarian malignancies. Materials and Methods We injected SK-OV-3 ovarian cancer cells peritoneally into nude mice to mimic human ovarian tumor metastasis. Overexpression of MUC13 mRNA was detected in metastatic implants from the xenografts by expression microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The DNA methylation status within the MUC13 promoter region was determined using bisulfite sequencing PCR and quantitative methylation-specific PCR. We evaluated the effects of exogenous MUC13 on cell invasion and migration using in vitro transwell assays. Results MUC13 mRNA expression was up-regulated, and methylation of specific CpG sites within the promoter was reduced in the metastatic implants relative to those in wild-type SK-OV-3 cells. Addition of a DNA methyltransferase inhibitor to SK-OV-3 cells induced MUC13 expression, thereby implying epigenetic regulation of MUC13 by promoter methylation. MUC13 overexpression increased migration and invasiveness, compared to control cells, suggesting aberrant up-regulation of MUC13 is strongly associated with progression of aggressive behaviors in ovarian cancer. Conclusion We provide novel evidence for epigenetic regulation of MUC13 in ovarian cancer. We suggest that the DNA methylation status within the MUC13 promoter region may be a potential biomarker of aggressive behavior in ovarian cancer.

The metastatic properties of cancer cells result from genetic and epigenetic alterations that lead to the abnormal expression of key genes regulating tumor phenotypes. Recent discoveries suggest that aberrant DNA methylation provides cancer cells with advanced metastatic properties; however, the precise regulatory mechanisms controlling metastasis-associated genes and their roles in metastatic transformation are largely unknown. We injected SK-OV-3 human ovarian cancer cells into the perineum of nude mice to generate a mouse model that mimics human ovarian cancer metastasis. We analyzed the mRNA expression and DNA methylation profiles in metastasized tumor tissues in the mice. The pro-oncogenic anterior gradient 2 (AGR2) gene showed increased mRNA expression and hypomethylation at CpG sites in its promoter region in the metastatic tumor tissues compared with the cultured SK-OV-3 cells. We identified crucial cytosine residues at CpG sites in the AGR2 promoter region. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine reduced the level of CpG methylation in the AGR2 promoter and increased the level of AGR2 expression. Next, we explored the functional role of AGR2 in the metastatic transformation of SK-OV-3 cells. SK-OV-3 cells overexpressing AGR2 showed increased migratory and invasive activity. Our results indicate that DNA methylation within the AGR2 promoter modulates more aggressive cancer cell phenotypes.

A lack of reliable biomarkers for the early detection and risk of metastatic recurrences makes ovarian cancer the most lethal gynecological cancer. To understand the molecular mechanisms involved in ovarian cancer metastasis in vivo, we analyzed the transcriptional expression pattern in metastatic implants of human ovarian carcinoma xenografts in mice. The expression of 937 genes was significantly different, by at least 2-fold, in the xenografts compared with that in SK-OV-3 cells. We investigated the mechanisms that regulate the expression of one of the profoundly upregulated genes, interferon-induced transmembrane protein 1 (IFITM1), in the metastatic implants. Specific CpG sites within the IFITM1 promoter were hypomethylated in the metastatic implants relative to those in the wild-type SK-OV-3 cells. Treating wild-type SK-OV-3 cells with the demethylating agent 5-aza-2'-deoxycytidine enhanced IFITM1 expression in a dose-dependent manner, implying transcriptional regulation by promoter methylation. We also found that IFITM1 overexpression caused increased migration and invasiveness in SK-OV-3 cells. Our results demonstrate that IFITM1 could be a novel metastasis-promoting gene that enhances the metastatic phenotype in ovarian cancer via epigenetic transcriptional regulation. Our findings also suggest that the status of DNA methylation within the IFITM1 promoter region could be a biomarker indicating metastatic progression in ovarian cancer.

OBJECTIVE The aim of the present study was to examine the expression of FOXP3, interleukin (IL)-10, transforming growth factor (TGF)-β1, IL-17A, and T helper 17 (TH17) cells/FOXP3+ regulatory T (Treg) cells balance in the gastric mucosa of children with Helicobacter pylori infection, in relation to the gastric histopathology. METHODS Antral mucosal biopsies were obtained from 20 children with H pylori(+) gastritis and 20 age- and sex-matched normal controls. Histopathology was assessed by the updated Sydney classification. Gene expression of FOXP3, IL-10, and TGF-β1 was analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining for FOXP3+ Treg and TH17 cells was performed. RESULTS The gene expression levels of FOXP3, TGF-β1, and IL-10 messenger RNA (mRNA) and the number of FOXP3+ Treg were significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.01). FOXP3 mRNA levels were correlated positively with TGF-β1 and IL-10 mRNA levels in the H pylori(+) gastritis group (P < 0.05). Furthermore, FOXP3 mRNA levels were correlated positively with the bacterial density, infiltration of polymorphonuclear cells, and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). The number of TH17 cells was significantly higher in the H pylori(+) gastritis group than in the control group (P < 0.05). In addition, the number of TH17 cells was correlated negatively with the bacterial density and positively with the inflammatory scores of polymorphonuclear cells and mononuclear cells in the H pylori(+) gastritis group (P < 0.05). A negative correlation between the TH17 cells/FOXP3+ Treg ratio and the bacterial density was demonstrated in the H pylori(+) gastritis group (P < 0.05). CONCLUSIONS This study suggested that a TH17/Treg balance toward a Treg-biased response favors the persistence of bacteria, causing chronic active gastritis.

Malignant gliomas are the most common and fatal brain tumors in adults. In particular, the strong invasiveness of glioma cells into the normal brain tissue makes eradication of glioma very difficult. Matrix metalloproteinases (MMPs) play a pivotal role in glioma invasion, and thus controlling MMP expression has been suggested as an important therapeutic target for brain tumors. In the present study, we investigated the effect of protopanaxatriol ginsenoside Rh1 on MMP expressions in human astroglioma U87MG and U373MG cells. RT-PCR analysis showed that Rh1 inhibits the mRNA expressions of MMP-1, -3, and -9 in PMA-stimulated U87MG and U373MG cells. Rh1 also suppressed the promoter activities of MMP-1, -3 and -9. The ELISA, Western blot, and zymographic analyses revealed that Rh1 inhibits the protein expression and/or enzymatic activity of MMP-1, -3 and -9. In accordance with the strong inhibitory effects of Rh1 on MMPs, Rh1 efficiently inhibited the invasion and migration of U87MG and U373MG glioma cells as demonstrated by Matrigel invasion assay and wound healing assay. Further mechanistic studies revealed that Rh1 inhibits MAPK and PI3K/Akt signaling pathways and downstream transcription factors such as NF-κB and AP-1, which play an important role in MMP gene expressions. The data collectively suggest that ginsenoside Rh1 may have a therapeutic potential for malignant gliomas.

p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin-reuptake inhibitors (SSRIs). Here we have identified SMARCA3, a chromatin-remodeling factor, as a novel target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. In response to the SSRI, fluoxetine, the expression of p11 is induced in both cell types, and the amount of the ternary complex of p11/annexin A2/SMARCA3 is increased. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway, and suggest a novel approach to the development of improved antidepressant therapies.

Metastasis is a major cause of therapeutic failure in ovarian cancer. To elucidate molecular mechanisms of ovarian cancer metastasis, we previously established a metastatic xenograft mouse model using human ovarian carcinoma SK-OV-3 cells. Using gene expression profiling, we found that γ-aminobutyric acid (GABA)A receptor π subunit (GABRP) expression was upregulated (>4-fold) in metastatic tissues from our xenograft mice compared with SK-OV-3 cells. Importantly, GABRP knockdown diminished the migration and invasion of SK-OV-3 cells, and reduced extracellular signal-regulated kinase (ERK) activation while overexpression of GABRP exhibited significantly increased cell migration, invasion and ERK activation. Moreover, treatment with the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126 similarly suppressed the migration and invasion of SK-OV-3 cells, implying that GABRP promotes these cellular behaviors by activating the MAPK/ERK pathway. Using genome-wide DNA methylation profiling, we identified hypomethylated CpG sites in the GABRP promoter in metastatic tissues from the xenograft mice compared with SK-OV-3 cells. Treatment with a DNA methyltransferase inhibitor demonstrated that methylation at −963 bp from the GABRP transcription start site (−963 CpG site) was critical for the epigenetic regulation of GABRP. Finally, we analyzed human ovarian cancer patient samples and showed DNA hypomethylation at the GABRP −963 CpG site in advanced stage, but not early-stage, primary tumors compared with their paired normal tissues. These findings suggest that GABRP enhances the aggressive phenotype of ovarian cancer cells, and that the DNA methylation status of the GABRP −963 CpG site may be useful for predicting the metastatic potential in ovarian cancer patients. A neurotransmitter-binding protein may play a prominent role in aggressive metastatic growth in ovarian cancer. Most patients with ovarian cancer are diagnosed late, and have a high risk of metastatic growth and a poor overall prognosis. A research team led by Jung-Hyuck Ahn and Woong Ju at Ewha Womans University in Seoul, Korea, have developed a mouse model for ovarian cancer that allowed them to home in on a potentially useful biomarker for metastasis. They identified a protein that forms part of the receptor for GABA, an important neurotransmitter, and demonstrated that it appeared to mediate invasive growth of tumor cells in mice. The researchers identified chemical tags on the gene encoding this protein that appear to regulate its production. Their preliminary analysis of human tumor samples suggests that these modifications may identify high-risk tumors.

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation and are thus a valuable source for the replacement of diseased or damaged organs. Previously, we reported that the tonsils can be an excellent reservoir of MSCs for the regeneration of skeletal muscle (SKM) damage. However, the mechanisms involved in the differentiation from tonsil-derived MSCs (T-MSCs) to myocytes via myoblasts remain unclear. To clarify these mechanisms, we analyzed gene expression profiles of T-MSCs during differentiation into myocytes compared with human skeletal muscle cells (hSKMCs). Total RNA was extracted from T-MSCs, T-MSC-derived myoblasts and myocytes, and hSKMCs and was subjected to analysis using a microarray. Microarray analysis of the three phases of myogenic differentiation identified candidate genes associated with myogenic differentiation. The expression pattern of undifferentiated T-MSCs was distinguishable from the myogenic differentiated T-MSCs and hSKMCs. In particular, we selected FNBP1L, which among the upregulated genes is essential for antibacterial autophagy, since autophagy is related to SKM metabolism and myogenesis. T-MSCs differentiated toward myoblasts and skeletal myocytes sequentially, as evidenced by increased expression of autophagy-related markers (including Beclin-1, LC3B and Atg5) and decreased expression of Bcl-2. Furthermore, we reconfirmed that autophagy has an effect on the mechanism of skeletal myogenic differentiation derived from T-MSCs by treatment with 5-azacytidine and bafilomycin A1. These data suggest that the transcriptome of the T-MSC-derived myocytes is similar to that of hSKMCs, and that autophagy has an important role in the mechanism of myogenic differentiation of T-MSCs.

To identify epigenetically regulated genes involved in the pathogenesis of Alzheimer's disease (AD) we analyzed global mRNA expression and methylation profiles in amyloid precursor protein (APP)-Swedish mutant-expressing AD model cells, H4-sw and selected heme oxygenase-1 (HMOX1), which is associated with pathological features of AD such as neurofibrillary tangles and senile plaques. We examined the epigenetic regulatory mechanism of HMOX1 and its application as a diagnostic and prognostic biomarker for AD. Our results show that HMOX1 mRNA and protein expression was approximately 12.2-fold and 7.9-fold increased in H4-sw cells, respectively. Increased HMOX1 expression was also detected in the brain, particularly the hippocampus, of AD model transgenic mice. However, the methylation of specific CpG sites within its promoter, particularly at CpG located -374 was significantly decreased in H4-sw cells. Treatment of neuroglioma cells with the demethylating agent 5-aza-2'-deoxycytidine resulted in reduced methylation of HMOX1 promoter accompanied by enhanced HMOX1 expression strongly supporting DNA methylation-dependent transcriptional regulation of HMOX1. Toxic Aβ-induced aberrant hypomethylation of HMOX1 at -374 promoter CpG site was correlated with increased HMOX1 expression. In addition to neuroglioma cells, we also found Aβ-induced epigenetic regulation of HMOX1 in human T lymphocyte Jurkat cells. We evaluated DNA methylation status of HMOX1 at -374 promoter CpG site in blood samples from AD patients, patients with mild cognitive impairment (MCI), and control individuals using quantitative methylation-specific polymerase chain reaction. We observed lower methylation of HMOX1 at the -374 promoter CpG site in AD patients compared to MCI and control individuals, and a correlation between Mini-Mental State Examination score and demethylation level. Receiver operating characteristics analysis revealed good discrimination of AD patients from MCI patients and control individuals. Our findings suggest that the methylation status of HMOX1 at a specific promoter CpG site is related to AD progression.

The Swedish mutation (K595N/M596L) of amyloid precursor protein (APP-swe) has been known to increase abnormal cleavage of cellular APP by Beta-secretase (BACE), which causes tau protein hyperphosphorylation and early-onset Alzheimer's disease (AD). Here, we analyzed the effect of APP-swe in global gene expression using deep transcriptome sequencing technique. We found 283 genes were down-regulated and 348 genes were up-regulated in APP-swe expressing H4-swe cells compared to H4 wild-type cells from a total of approximately 74 million reads of 38 base pairs from each transcriptome. Two independent mechanisms such as kinase and phosphatase signaling cascades leading hyperphosphorylation of tau protein were regulated by the expression of APP-swe. Expressions of catalytic subunit as well as several regulatory subunits of protein phosphatases 2A were decreased. In contrast, expressions of tau-phosphorylating glycogen synthase kinase 3ß (GSK-3ß), cyclin dependent kinase 5 (CDK5), and cAMP-dependent protein kinase A (PKA) catalytic subunit were increased. Moreover, the expression of AD-related Aquaporin 1 and presenilin 2 expression was regulated by APP-swe. Taken together, we propose that the expression of APP-swe modulates global gene expression directed to AD pathogenesis.

The actions of LH to induce ovulation and luteinization of preovulatory follicles are mediated principally by activation of cAMP-dependent protein kinase (PKA) in granulosa cells. PKA activity is targeted to specific locations in many cells by A kinase-anchoring proteins (AKAPs). We previously showed that FSH induces expression of microtubule-associated protein (MAP) 2D, an 80-kDa AKAP, in rat granulosa cells, and that MAP2D coimmunoprecipitates with PKA-regulatory subunits in these cells. Here we report a rapid and targeted dephosphorylation of MAP2D at Thr256/Thr259 after treatment with human chorionic gonadotropin, an LH receptor agonist. This event is mimicked by treatment with forskolin or a cAMP analog and is blocked by the PKA inhibitor myristoylated-PKI, indicating a role for cAMP and PKA signaling in phosphoregulation of granulosa cell MAP2D. Furthermore, we show that Thr256/Thr259 dephosphorylation is blocked by the protein phosphatase 2A (PP2A) inhibitor, okadaic acid, and demonstrate interactions between MAP2D and PP2A by coimmunoprecipitation and microcystin-agarose pull-down. We also show that MAP2D interacts with glycogen synthase kinase (GSK) 3beta and is phosphorylated at Thr256/Thr259 by this kinase in the basal state. Increased phosphorylation of GSK3beta at Ser9 and the PP2A B56delta subunit at Ser566 is observed after treatment with human chorionic gonadotropin and appears to result in LH receptor-mediated inhibition of GSK3beta and activation of PP2A, respectively. Taken together, these results show that the phosphorylation status of the AKAP MAP2D is acutely regulated by LH receptor-mediated modulation of kinase and phosphatase activities via PKA.

Article; International audience; Dopamine orchestrates motor behaviour and reward-driven learning. Perturbations of dopamine signalling have been implicated in several neurological and psychiatric disorders, and in drug addiction. The actions of dopamine are mediated in part by the regulation of gene expression in the striatum, through mechanisms that are not fully understood. Here we show that drugs of abuse, as well as food reinforcement learning, promote the nuclear accumulation of 32-kDa dopamine-regulated and cyclic-AMP-regulated phosphoprotein (DARPP-32). This accumulation is mediated through a signalling cascade involving dopamine D1 receptors, cAMP-dependent activation of protein phosphatase-2A, dephosphorylation of DARPP-32 at Ser 97 and inhibition of its nuclear export. The nuclear accumulation of DARPP-32, a potent inhibitor of protein phosphatase-1, increases the phosphorylation of histone H3, an important component of nucleosomal response. Mutation of Ser 97 profoundly alters behavioural effects of drugs of abuse and decreases motivation for food, underlining the functional importance of this signalling cascade

Ovarian cancer (OC) metastasis has unique biological behavior and most commonly occurs via the transcoelomic route. Previously, we established a mouse xenograft model of human ovarian carcinoma and analyzed alterations in gene expression during metastasis. Among the genes that were differentially expressed more than 2-fold in the xenografts compared with the SK-OV-3 cells, we selected synaptotagmin-like protein 2 (SYTL2) and investigated the mechanisms regulating its expression and its gene function in OC. The mRNA expression of SYTL2 was significantly upregulated and the methylation of specific CpG sites within the SYTL2 promoter was decreased in the metastatic implants from the ovarian carcinoma xenografts compared to wild-type SK-OV-3 cells. Treatment with the demethylating agent 5-aza‑2'-deoxycytidine and/or the histone deacetylase inhibitor Trichostatin A induced upregulation of SYTL2 in SK-OV-3 cells, implying that a DNA methylation-dependent epigenetic mechanism is involved in the regulation of SYTL2 expression. We also found that overexpression of SYTL2 promoted metastatic potential, including increased migration and invasiveness in the ovarian carcinoma cells. Furthermore, we utilized publicly available gene expression data to confirm the correlation between SYTL2 expression and poor prognosis in serous-type OC patients. Our findings provide novel evidence for the direct association of SYTL2 with the metastatic potential of ovarian carcinoma cells and its influence on metastatic recurrence of OC.

Our previous studies of DARPP-32 in striatal slices have shown that activation of D1 receptors leads to cAMP-dependent dephosphorylation of Thr-75, the Cdk5 site in DARPP-32. In the current study, we have elucidated a mechanism whereby protein phosphatase 2A (PP2A) is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of Thr-75. PP2A consists of a catalytic C subunit that associates with the scaffolding A subunit and a variety of B subunits. We have found that the A/C subunits of PP2A, in association with the B56δ (or PPP2R5D) regulatory subunit, is an active DARPP-32 phosphatase. The B56δ subunit expressed in HEK293 cells forms a heterotrimeric assembly that catalyzes PKA-mediated dephosphorylation at Thr-75 in DARPP-32 (also cotransfected into HEK293 cells). The B56δ subunit is phosphorylated by PKA, and this increases the overall activity of PP2A in vitro and in vivo . Among four PKA-phosphorylation sites identified in B56δ in vitro , Ser-566 was found to be critical for the regulation of PP2A activity. Moreover, Ser-566 was phosphorylated by PKA in response to activation of D1 receptors in striatal slices. Based on these studies, we propose that the B56δ/A/C PP2A complex regulates the dephosphorylation of DARPP-32 at Thr-75, thereby helping coordinate the efficacy of dopaminergic neurotransmission in striatal neurons. Moreover, stimulation of protein phosphatase activity by this mechanism may represent an important signaling pathway regulated by cAMP in neurons and other types of cell.

Although cisplatin is one of the most effective antitumor drugs for ovarian cancer, the emergence of chemoresistance to cisplatin in over 80% of initially responsive patients is a major barrier to successful therapy. The precise mechanisms underlying the development of cisplatin resistance are not fully understood, but alteration of DNA methylation associated with aberrant gene silencing may play a role. To identify epigenetically regulated genes directly associated with ovarian cancer cisplatin resistance, we compared the expression and methylation profiles of cisplatin-sensitive and -resistant human ovarian cancer cell lines. We identified α-N-acetylgalactosaminidase (NAGA) as one of the key candidate genes for cisplatin drug response. Interestingly, in cisplatin-resistant cell lines, NAGA was significantly downregulated and hypermethylated at a promoter CpG site at position +251 relative to the transcriptional start site. Low NAGA expression in cisplatin-resistant cell lines was restored by treatment with a DNA demethylation agent, indicating transcriptional silencing by hyper-DNA methylation. Furthermore, overexpression of NAGA in cisplatin-resistant lines induced cytotoxicity in response to cisplatin, whereas depletion of NAGA expression increased cisplatin chemoresistance, suggesting an essential role of NAGA in sensitizing ovarian cells to cisplatin. These findings indicate that NAGA acts as a cisplatin sensitizer and its gene silencing by hypermethylation confers resistance to cisplatin in ovarian cancer. Therefore, we suggest NAGA may be a promising potential therapeutic target for improvement of sensitivity to cisplatin in ovarian cancer. Citation Format: Woong Ju, Hye Youn Sung, Jung-Hyuck Ahn. Epigenetic modification of α-N-acetylgalactosaminidase (NAGA) enhances cisplatin resistance in ovarian cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A171.

An increase in amyloid-β (Aβ) production is a major pathogenic mechanism associated with Alzheimer's disease (AD), but little is known about possible homeostatic control of the amyloidogenic pathway. Here we report that the amyloid precursor protein (APP) intracellular domain (AICD) downregulates Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1 or WASF1) as part of a negative feedback mechanism to limit Aβ production. The AICD binds to the Wasf1 promoter, negatively regulates its transcription and downregulates Wasf1 mRNA and protein expression in Neuro 2a (N2a) cells. WAVE1 interacts and colocalizes with APP in the Golgi apparatus. Experimentally reducing WAVE1 in N2a cells decreased the budding of APP-containing vesicles and reduced cell-surface APP, thereby reducing the production of Aβ. WAVE1 downregulation was observed in mouse models of AD. Reduction of Wasf1 gene expression dramatically reduced Aβ levels and restored memory deficits in a mouse model of AD. A decrease in amounts of WASF1 mRNA was also observed in human AD brains, suggesting clinical relevance of the negative feedback circuit involved in homeostatic regulation of Aβ production.

To test whether resveratrol provides benefits via estrogen receptors (ERs) in the blood-brain barrier of estrogen-deficient females, ovariectomized mice were treated with resveratrol then were subjected to transient middle cerebral artery occlusion (MCAO). Compared with vehicle treatment, resveratrol reduced infarct volume and neurologic deficits after MCAO. Basal tight junction (TJ) protein levels in the brain were increased by resveratrol. After MCAO, blood-brain barrier breakdown reduced levels of TJ proteins, and induction of HIF-1α and VEGF were attenuated by resveratrol. These effects were reversed by the ERs antagonist, ICI182,780. In mouse brain, endothelial cells (bEnd.3) exposed to hypoxia, resveratrol treatment protected the cells against cytotoxicity, increases of paracellular permeability and changes in levels of TJ protein and HIF-1α/VEGF proteins. These effects were reversed by ICI182,780 but not by specific ERα or ERβ antagonists, indicating nonspecific ER mediated effects. Altogether, these results showed that neuroprotective effects of resveratrol in ovariectomized mice were mediated by ERs and associated with tightening of blood-brain barrier, suggesting that resveratrol can be an alternative to estrogens to protect the brains of estrogen-deficient females against ischemic insult.

Caveolae-like domains (CLDs) have been hypothesized to mediate apoptosis, since they contain sphingomyelin and initiate the conversion of sphingomyelin to ceramide. To address whether CLDs are directly involved in apoptosis, CLDs from U937 cells were isolated, taking advantage of their detergent insolubility and low density. The CLDs contained alkaline phosphatase as well as many signaling molecules, including Fyn, protein kinase Cα, Raf-1, phospholipase Cγ1, and tyrosine phosphoproteins. Immunoblotting and immunofluorescent data showed that TNF receptor 1 colocalized with CD36 in CLDs, suggesting that TNF-α-initiated apoptosis occurs in CLDs. When cells were incubated with lipoprotein-deficient medium, the cholesterol concentration was greatly decreased in CLDs but not in other fractions, implying that the CLDs were selectively disrupted. In the CLD-disrupted cells, the surface expression of TNF receptor 1 and CD36 was significantly reduced. Analysis of cellular morphology, percent DNA fragmentation, DNA laddering, and caspase-3 activity showed that TNF-α-mediated apoptosis was blocked in CLD-disrupted cells, whereas anti-Fas-mediated apoptosis was not. Since Fas was not found in CLDs of Jurkat cells, apoptosis by Fas ligation might not require CLDs. Taken together, these data strongly imply that TNF-α-mediated apoptosis is initiated in CLDs.

Granulocyte-macrophage colony stimulating factor (GM-CSF) induces proliferation and maturation of myeloid progenitor cells and also activates neutrophils. In order to investigate the pleiotropic effects of GM-CSF stimulation, we examined the signaling pathways of protein tyrosine kinases (PTKs) and signal transducers and activators of transcription (STATs) in GM-CSF-dependent proliferation of leukemia cells. Using TF-1, a GM-CSF-dependent human erythroleukemia cell line, we found that GM-CSF enhanced DNA-binding and tyrosine phosphorylation of STAT3. GM-CSF receptor (GM-CSFR) and c-Fes tyrosine kinase were also activated upon GM-CSF stimulation. Furthermore, c-Fes formed a complex with STAT3. Experiments using a c-Fes mutant that lacked tyrosine kinase activity revealed that the activation of STAT3 is kinase-dependent, but that the c-Fes-STAT3 interaction is not affected by c-Fes tyrosine kinase activity. The results suggest that STAT3 is activated by c-Fes tyrosine kinase through direct interaction during hematopoietic cell proliferation induced by GM-CSF.

Swedish double mutation (KM670/671NL) of amyloid precursor protein (APP) is reported to increase toxic amyloid b (Ab) production via aberrant cleavage at the beta-secretase site and thereby cause early-onset Alzheimer's disease (AD). However, the underlying molecular mechanisms leading to AD pathogenesis remains largely unknown. Previously, our transcriptome sequence analyses revealed global expressional modifications of over 600 genes in APP-Swedish mutant-expressing H4 (H4sw) cells compared to wild type H4 cells. Insulin-like growth factor binding protein 3 (IGFBP3) is one gene that showed significantly decreased mRNA expression in H4-sw cells. In this study, we investigated the functional role of IGFBP3 in AD pathogenesis and elucidated the mechanisms regulating its expression. We observed decreased IGFBP3 expression in the H4-sw cell line as well as the hippocampus of AD model transgenic mice. Treatment with exogenous IGFBP3 protein inhibited A beta(1-42)-induced cell death and caspase-3 activity, whereas siRNA-mediated suppression of IGFBP3 expression induced cell death and caspase-3 cleavage. In primary hippocampal neurons, administration of IGFBP3 protein blocked apoptotic cell death due to A beta(1-42) toxicity. These data implicate a protective role for IGFBP3 against A beta(1-42)-mediated apoptosis. Next, we investigated the regulatory mechanisms of IGFBP3 expression in AD pathogenesis. We observed abnormal IGFBP3 hypermethylation within the promoter CpG island in H4-sw cells. Treatment with the DNA methyltransferase inhibitor 5-aza-29-deoxycytidine restored IGFBP3 expression at both the mRNA and protein levels. Chronic exposure to A beta(1-42) induced IGFBP3 hypermethylation at CpGs, particularly at loci 2164 and 2173, and subsequently suppressed IGFBP3 expression. Therefore, we demonstrate that expression of anti-apoptotic IGFBP3 is regulated by epigenetic DNA methylation, suggesting a mechanism that contributes to AD pathogenesis.

Purpose Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. Materials and methods Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. Results SLC6A12 expression was 8.1-14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41-62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. Conclusion Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.

Liquid-forming impurities (CaO + SiO2) were placed on the top of undoped and MgO-doped Al2O3 powder compacts, and the microstructural evolution and the extent of liquid penetration during heat treatment were investigated. Compared with the undoped specimen, abnormal grain growth was limited and the impurities penetrated more deeply in the MgO-doped specimen. The homogenization of the microstructure and the enhanced liquid penetration in MgO-doped specimens could be explained in terms of an interface roughening or a modification of the liquid-(glass-) phase characteristics, such as viscosity.

Alumina specimens with various amounts of CaO and SiO2 (1:2 ratio) were prepared, and their abnormal grain growth (AGG) kinetics were investigated. A plot of the area fraction covered by abnormal grains versus log (sintering time) had a sigmoidal shape with an apparent incubation period before the onset of AGG. The overall kinetics of AGG was similar to that of a phase transformation controlled by nucleation and growth. The incubation time and the end point of AGG were strongly dependent on the amount of liquid-forming additives. Correspondingly, the final microstructure was affected by the liquid content: a large grain size and a high aspect ratio at low liquid content and a small grain size and a low aspect ratio at high liquid content.

We previously showed that all-trans retinoic acid (atRA) decreased nitric oxide (NO) production through Akt-mediated decreased phosphorylation of endothelial NO synthase at serine 1179 (eNOS-Ser1179) in bovine aortic endothelial cells (BAEC). Since protein phosphatase 2A (PP2A) was also reported to decrease eNOS-Ser1179 phosphorylation, we investigated using BAEC whether PP2A mediates atRA-induced eNOS-Ser1179 dephosphorylation and subsequent decreased NO production. Treatment with okadaic acid (5 nM), a selective PP2A inhibitor, or ectopic expression of small interference RNA (siRNA) of PP2A catalytic subunit α (PP2A Cα) significantly increased eNOS-Ser1179 phosphorylation and NO production. Each treatment also significantly reversed atRA-induced observed effects, suggesting a role for PP2A. We also found that atRA significantly increased cellular PP2A activity. However, Western blot analysis revealed that atRA did not increase the expression of PP2A Cα, although it significantly increased the level of B56α of PP2A regulatory B subunit (PP2A B56α), but not PP2A B55α and PP2A B56δ. Real-time PCR assay confirmed a significant increase in PP2A B56α mRNA expression in atRA-treated cells. Ectopic expression of siRNA of PP2A B56α significantly reversed atRA-induced inhibitory effects on eNOS-Ser1179 phosphorylation and NO production, suggesting a role for PP2A B56α. Our study demonstrates for the first time that atRA decreases eNOS-Ser1179 phosphorylation and NO release at least in part by increasing PP2A B56α-mediated PP2A activity in BAEC.

The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer’s disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterations in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the −435, −295, and −271 CpG sites of CTIF, and at the −505 to −341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at −432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.

To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines.mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay.From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P = 4.38 x 10(-7)) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway.Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.

To characterize the biochemical properties of the PP2A regulatory B subunit, PPP2R5D, we analyzed its phosphorylation sites, stoichiometry and effect on holoenzyme activity. PPP2R5D was phosphorylated on Ser-53, Ser-68, Ser-81, and Ser-566 by protein kinase A, and mutations at all four of these sites abolished any significant phosphorylation in vitro. In HEK293 cells, however, the Ser-566 was the major phosphorylation site after PKA activation by forskolin, with marginal phosphorylation on Ser-81. Inhibitory tyrosine phosphorylation on Tyr-307 of the PP2A catalytic C subunit was decreased after forskolin treatment. Kinetic analysis showed that overall PP2A activity was increased with phosphorylation by PPP2R5D phosphorylation. The apparent Km was reduced from 11.25 microM to 1.175 microM with PPP2R5D phosphorylation, resulting in an increase in catalytic activity. These data suggest that PKAmediated activation of PP2A is enabled by PPP2R5D phosphorylation, which modulates the affinity of the PP2A holoenzyme to its physiological substrates.

In dopaminoceptive neurons, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in integrating the effects of dopamine and other neurotransmitters. Phosphorylation of DARPP-32 at Thr-34 by protein kinase A results in inhibition of protein phosphatase 1 (PP1), and phosphorylation at Thr-75 by Cdk5 (cyclin-dependent kinase 5) results in inhibition of protein kinase A. Dephosphorylation at Thr-34 involves primarily the Ca 2+ -dependent protein phosphatase, PP2B (calcineurin), whereas dephosphorylation of Thr-75 involves primarily PP2A, the latter being subject to control by both cAMP- and Ca 2+ -dependent regulatory mechanisms. In the present study, we have investigated the mechanism of Ca 2+ -dependent regulation of Thr-75 by PP2A. We show that the PR72 (or B″ or PPP2R3A) regulatory subunit of PP2A is highly expressed in striatum. Through the use of overexpression and down-regulation by using RNAi, we show that PP2A, in a heterotrimeric complex with the PR72 subunit, mediates Ca 2+ -dependent dephosphorylation at Thr-75 of DARPP-32. The PR72 subunit contains two Ca 2+ binding sites formed by E and F helices (EF-hands 1 and 2), and we show that the former is necessary for the ability of PP2A activity to be regulated by Ca 2+ , both in vitro and in vivo . Our studies also indicate that the PR72-containing form of PP2A is necessary for the ability of glutamate acting at α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and NMDA receptors to regulate Thr-75 dephosphorylation. These studies further our understanding of the complex signal transduction pathways that regulate DARPP-32. In addition, our studies reveal an alternative intracellular mechanism whereby Ca 2+ can activate serine/threonine phosphatase activity.