Down-Regulated CUEDC2 Increases GDNF Expression by Stabilizing CREB Through Reducing Its Ubiquitination in Glioma.

Abnormally high expression of glial cell line-derived neurotrophic factor (GDNF) derived from glioma cells has essential impacts on gliomagenesis and development, but the molecular basis underlying increased GDNF expression in glioma cells remain unclear. This work aimed to study the molecular mechanisms that may explain the accumulation of GDNF in glioma. Firstly, we observed that cAMP response element-binding protein (CREB), known as an important transcription factor for binding of GDNF promoter region, was highly expressed with an apparent accumulation into the nucleus of glioma cells, which may contribute to the transcription of GDNF. Secondly, CUE domain-containing protein 2 (CUEDC2), a ubiquitin-regulated protein, could increase the amount of binding between the E3 ligase tripartite motif-containing 21 (TRIM21) and CREB and affect the CREB level. Like our previous study, it showed that there was a significantly down-regulation of CUEDC2 in glioma. Finally, our data suggest that GDNF expression is indirectly regulated by transcription factor ubiquitination. Indeed, down-regulation of CUEDC2, decreased the ubiquitination and degradation of CREB, which was associated to high levels of GDNF. Furthermore, abundant CREB involved in the binding to the GDNF promoter region contributes to GDNF high expression in glioma cells. Collectively, it was verified the GDNF expression was affected by CREB ubiquitination regulated by CUEDC2 level.

Author Correction: Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model.

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MiRNAs Mediate GDNF-Induced Proliferation and Migration of Glioma Cells.

BACKGROUND/AIMS: Glial cell line-derived neurotrophic factor (GDNF) is an important factor promoting invasive glioma growth. This study was performed to reveal a unique mechanism of glioma cell proliferation and migration. METHODS: Human U251 glioma cells were used to screen the optimal GDNF concentration and treatment time to stimulate proliferation and migration. MicroRNA (MiRNA) expression profiles were detected by microarray and confirmed by real-time polymerase chain reaction (PCR). The target genes of differentially expressed miRNAs were predicted by miRWalk, and those targeted by multiple miRNAs were screened with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A regulatory miRNA network was constructed using ingenuity pathway analysis (IPA). Target gene expression of differentially expressed miRNAs was examined by real-time PCR or mRNA microarray. RESULTS: The results show that 50 ng/mL GDNF for 24 h significantly promotes U251 glioma cell proliferation and migration (P < 0.05). Seven miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-629-5p, hsa-miR-3609, hsa-miR-183-5p, and hsa-miR-487b-3p) were significantly up-regulated after GDNF treatment (P < 0.05). These miRNAs are primarily involved in signal transduction, cell adhesion and cell cycle through mitogen-activated protein kinase (MAPK) signaling, focal adhesion and glioma signal pathways. Five of these miRNAs (hsa-miR-194-5p, hsa-miR-152-3p, hsa-miR-205-5p, hsa-miR-183-5p, and hsa-miR-487b-3p) co-regulate TP53 and Akt. mRNA expression levels of four genes co-targeted by two or more up-regulated miRNAs were significantly decreased after GDNF treatment (P < 0.05). CONCLUSION: GDNF treatment of U251 glioma cells significantly increased the expression of seven miRNAs involved in cell adhesion and the cell cycle.

Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model.

Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

Egr-1 and RNA POL II facilitate glioma cell GDNF transcription induced by histone hyperacetylation in promoter II.

The specific mechanisms for epigenetic regulation of gene transcription remain to be elucidated. We previously demonstrated that hyperacetylation of histone H3K9 in promoter II of glioma cells promotes high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene. This hyperacetylation significantly enhanced Egr-1 binding and increased the recruitment of RNA polymerase II (RNA POL II) to that region (P < 0.05). Egr-1 expression was abnormally increased in C6 glioma cells. Further overexpression of Egr-1 significantly increased Egr-1 binding to GDNF promoter II, while increasing RNA POL II recruitment, thus increasing GDNF transcription (P < 0.01). When the acetylation of H3K9 in the Egr-1 binding site was significantly reduced by the histone acetyltransferase (HAT) inhibitor curcumin, binding of Egr-1 to GDNF promoter II, RNA POL II recruitment, and GDNF mRNA expression were significantly downregulated (P < 0.01). Moreover, curcumin attenuated the effects of Egr-1 overexpression on Egr-1 binding, RNA POL II recruitment, and GDNF transcription (P < 0.01). Egr-1 and RNA POL II co-existed in the nucleus of C6 glioma cells, with overlapping regions, but they were not bound to each other. In conclusion, highly expressed Egr-1 may be involved in the recruitment of RNA POL II in GDNF promoter II in a non-binding manner, and thereby involved in regulating GDNF transcription in high-grade glioma cells. This regulation is dependent on histone hyperacetylation in GDNF promoter II.

Role of Autophagy in Capsaicin-Induced Apoptosis in U251 Glioma Cells.

In recent years, the role of capsaicin in cancer prevention and treatment has gained people's attention. However, the mechanism of anti-glioma cells by capsaicin has not been elucidated. Here, we discuss the mechanism of capsaicin in U251 cells. Cell viability was detected by MTT and extracellular LDH measurements, while immunofluorescence was performed to measure changes of LC3 in U251 cells. The expressions of LC3II, Puma-α, Beclin1, P62, Procaspase-3, and P53 were observed by immunoblotting. The cell viability decreased and the punctate patterns of LC3 in U251 cells were observed after Capsaicin treatment. Meanwhile, the expressions of Beclin1, P62, and Puma-α increased. After using 3-MA, the expressions of Beclin1 and Procaspase-3 were reduced while those of P53 and Puma-α increased. The expression of LC3II was increased after Pifithrin-α treatment. Therefore, we believed that capsaicin could induce apoptosis in U251 cells, and the inhibition of autophagy could contribute to apoptosis.

An Epigenetic Mechanism of High Gdnf Transcription in Glioma Cells Revealed by Specific Sequence Methylation.

Glioma cells express high levels of GDNF. When investigating its transcriptional regulation mechanism, we observed increased or decreased methylation of different cis-acting elements in the gdnf promoter II. However, it is difficult to determine the contributions of methylation changes of each cis-acting element to the abnormally high transcription of gdnf gene. To elucidate the contributions of methylation changes of specific cis-acting elements to the regulation of gdnf transcription, we combined gene site-directed mutation, molecular cloning, and dual luciferase assay to develop the "specific sequence methylation followed by plasmid recircularization" method to alter methylation levels of specific cis-acting elements in the gdnf promoter in living cells and assess gene transcriptional activity. This method successfully introduced artificial changes in the methylation of different cis-acting elements in the gdnf promoter II. Moreover, compared with unmethylated gdnf promoter II, both silencer II hypermethylation plus enhancer II unmethylation and hypermethylation of the entire promoter II (containing enhancer II and silencer II) significantly enhanced gdnf transcriptional activity (P < 0.05), and no significant difference was noted between these two hypermethylation patterns (P > 0.05). Enhancer II hypermethylation plus silencer II unmethylation did not significantly affect gene transcription (P > 0.05). Furthermore, we found significantly increased DNA methylation in the silencer II of the gdnf gene in high-grade astroglioma cells with abnormally high gdnf gene expression (P < 0.01). The absence of silencer II significantly increased gdnf promoter II activity in U251 cells (P < 0.01). In conclusion, our specific sequence methylation followed by plasmid recircularization method successfully altered the methylation levels of a specific cis-acting element in a gene promoter in living cells. This method allows in-depth investigation of the impact of methylation changes of different cis-acting elements in the same promoter on gene transcriptional activity. Our findings provide preliminary evidence that silencer II hypermethylation in the gdnf promoter II may underlie high gene transcription in high-grade glioma cells.

Design and synthesis of novel 1,2,3-triazole-pyrimidine hybrids as potential anticancer agents.

A series of novel 1,2,3-triazole-pyrimidine hybrids were designed, synthesized and evaluated for their anticancer activity against four selected cancer cell lines (MGC-803, EC-109, MCF-7 and B16-F10). Most of the synthesized compounds exhibited moderate to good activity against all the cancer cell lines selected. Compound 17 showed the most excellent anticancer activity with single-digit micromolar IC50 values ranging from 1.42 to 6.52 µM. Further mechanism studies revealed that compound 17 could obviously inhibit the proliferation of EC-109 cancer cells by inducing apoptosis and arresting the cell cycle at G2/M phase.

Egr-1 participates in abnormally high gdnf gene transcription mediated by histone hyperacetylation in glioma cells.

Abnormally high transcription of the glial cell-line derived neurotrophic factor (gdnf) gene in glioma cells is related to the hyperacetylation of histone H3 lysine 9 (H3K9) in its promoter region II, but the mechanism remains unclear. There are three consecutive putative binding sites for the transcription factor early growth response protein 1(Egr-1) in promoter region II of the gdnf gene, and Egr-1 participates in gdnf gene transcription activation. Here we show that the acetylation level of H3K9 at Egr-1 binding sites in gdnf gene promoter region II in rat C6 astroglioma cells was significantly higher than that in normal astrocytes, and the binding capacity was also significantly higher. In C6 astroglioma cells, gdnf gene transcription significantly decreased after Egr-1 knock-down. In addition, the deletion or mutation of the Egr-1 binding site also significantly down-regulated the activity of promoter region II of this gene in vitro. When curcumin decreased the acetylation level of H3K9 at the Egr-1 binding site, the binding of Egr-1 to promoter region II and GDNF mRNA levels significantly decreased. In contrast, trichostatin A treatment significantly increased H3K9 acetylation at the Egr-1 binding site, which significantly increased both the binding of Egr-1 with promoter region II and GDNF mRNA levels. In this context, knocking down Egr-1 significantly reduced the elevation in gdnf gene transcription. Collectively, our results demonstrate that the hyperacetylation of H3K9 at Egr-1 binding sites in promoter region II of the gdnf gene can up-regulate the binding of Egr-1 to increase gdnf gene transcription in glioma cells.

Hyperacetylation of histone H3K9 involved in the promotion of abnormally high transcription of the gdnf gene in glioma cells.

The mechanism underlying abnormally high transcription of the glial cell line-derived neurotrophic factor (GDNF) gene in glioma cells is not clear. In this study, to assess histone H3K9 acetylation levels in promoters I and II of the gdnf gene in normal human brain tissue, low- and high-grade glioma tissues, normal rat astrocytes, and rat C6 glioblastoma cells, we employed chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR), real-time PCR, and a pGL3 dual fluorescence reporter system. We also investigated the influence of treatment with curcumin, a histone acetyltransferase inhibitor, and trichostatin A (TSA), a deacetylase inhibitor, on promoter acetylation and activity and messenger RNA (mRNA) expression level of the gdnf gene in C6 cells. Compared to normal brain tissue, H3K9 acetylation in promoters I and II of the gdnf gene increased significantly in high-grade glioma tissues but not in low-grade glioma tissues. Moreover, H3K9 promoter acetylation level of the gdnf gene in C6 cells was also remarkably higher than in normal astrocytes. In C6 cells, curcumin markedly decreased promoter II acetylation and activity and GDNF mRNA expression. Conversely, all three measurements were significantly increased following TSA treatment. Our results suggest that histone H3K9 hyperacetylation in promoter II of the gdnf gene might be one of the reasons for its abnormal high transcription in glioma cells.

Synthesis and biological evaluation of dehydroepiandrosterone-fused thiazole, imidazo[2,1-b]thiazole, pyridine steroidal analogues.

A series of steroidal[17,16-d]thiazole, steroidal[1,2-b]pyridine and steroidal[17,16-d]thiazole[2,1-b]imidazo products were synthesized through a convenient and productive method. Anti-proliferation activity against EC109 (human esophageal carcinoma), EC9706 (human esophageal carcinoma) and MGC-803 (human gastric carcinoma) cell lines was examined in vitro. Among the screened compounds, several highly potential compounds were located.

Design and synthesis of novel D-ring fused steroidal heterocycles.

Using dehydroepiandrosterone as the starting material, we have synthesized a series of steroid analogs possessing a D-ring fused with heterocycles which are pyridine, imidazo [2,1-b]thiazoles or substituted thiazole imines. All the final structures are first reported and identified by NMR and MS spectroscopys, the yields of these products are moderate to good and the reaction conditions are mild. The cytotoxicity of the synthesized compounds against EC-109(human esophageal carcinoma), EC-9706(human esophageal carcinoma), MGC-803(human gastric carcinoma) were investigated.

[G protein-coupled receptor 3: a key factor in the regulation of the nervous system and follicle development].

G protein-coupled receptors (GPCRs), the largest cell surface receptor superfamily, are involved in many physiological and pathological processes. G protein-coupled receptor 3 (Gpr3) is a newly discovered sphingosine 1-phosphate receptor, which directly or indirectly takes part in regulating the processes of nervous system and follicle development in the vertebrates. As a potential therapeutic drug target for a variety of neurological diseases and premature ovarian failure, its physiological function and biological mechanisms deserve further studies. In this paper, we reviewed the functions of Gpr3 in the processes of nervous system development and ovarian follicular development in the vertebrates.

Changes in transcriptional factor binding capacity resulting from promoter region methylation induce aberrantly high GDNF expression in human glioma.

Glial cell line-derived neurotrophic factor (GDNF), which belongs to transforming growth factor ß superfamily, plays important roles in glioma pathogenesis. Gdnf mRNA is aberrantly increased in glioma cells, but the underlying transcription mechanism is unclear. Here, we found that although the base sequence in the promoter region of the gdnf gene was unchanged in glioma cells, there were significant changes in the methylation level of promoter region I (P < 0.05) in both high- and low-grade glioma tissues. However, the methylation degree in promoter region II was notably decreased in low-grade glioma tissue compared to normal brain tissue (P < 0.05), and the demethylation sites were mainly located in the enhancer region. Conversely, methylation was markedly increased in high-grade glioma tissue (P < 0.05), and the sites with decreased methylation level were mainly located in the silencer region. The binding capacities of several transcriptional factors, such as activating protein 2, specificity protein 1, ETS-related gene 2, and cAMP response element binding protein, which specifically bind to regions with altered methylation status decreased along with the pathological grade of glioma, and the differences between high-grade glioma and normal brain tissue were significant (P < 0.05). The results suggest that changes in transcriptional factor binding capacity are due to changes in promoter region methylation and might be the underlying mechanism for aberrantly high gdnf expression in glioma.

Facile synthesis of novel D-ring modified steroidal dienamides via rearrangement of 2H-pyrans.

A simple and practical method for synthesis of the D-ring modified steroidal dienamides (4a-k) from the steroidal α,α-dicyanoalkene 3 and aldehydes via vinylogous aldol reaction was first reported. By using NaOAc as a base, the desired products were obtained in moderate to good yields in ethanol under mild conditions. All the synthesized steroidal dienamides are new and are currently being evaluated for their biological activities.

Sphingosine 1-phosphate acts as an activator for the porcine Gpr3 of constitutively active G protein-coupled receptors.

We cloned the complete coding sequences of porcine Gpr3, Gpr6, and Gpr12 genes. Further, on the basis of their high levels of sequence similarity, these genes are identified as a subfamily of G protein-coupled receptors. These putative protein sequences also showed high sequence identity with other mammalian orthologs, including several highly conserved motifs. A wide expression of the Gpr3 gene in pigs was observed through tissue distribution analysis by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR, specially in the brain, pituitary, fat, liver and oocyte, where its strong expression was observed. The Gpr3 gene was found to be located on chromosome 6 and a single exon coded for the entire open-reading frame. Expression of porcine Gpr3 in HEK293 cells resulted in constitutive activation of adenylate cyclase (AC) similar in amplitude to that produced by fully stimulated G(s)-coupled receptors. Moreover, sphingosine 1-phosphate (S1P) could increase AC activation via the constitutively active Gpr3 receptor. When a Gpr3-green fluorescent protein (GFP) construct was expressed in HEK293 cells, GFP-labeled Gpr3 protein was shown to be localized in the plasmalemma and subcellular membranes. After S1P treatment, agonist-mediated internalization could be visualized by confocal microscopy. In short, our findings suggest the porcine Gpr3, Gpr6, and Gpr12 genes as a subfamily of G protein-coupled receptors, and porcine Gpr3 was a constitutively active G protein-coupled receptor. Constitutive activation of AC and agonist-mediated internalization of Gpr3 receptor could be modulated by the S1P, suggesting that S1P might act as an activator for porcine Gpr3 receptor.

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells.

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated 'gene silencing' of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.