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Morphological and functional changes in cells during the epithelial-mesenchymal transition (EMT) process are known to be regulated by alternative splicing. However, only a few splicing factors involved in EMT have been reported and their underlying mechanisms remain largely unknown. Here, we showed that an isoform of tight junction protein 1 (TJP1) lacking exon 20 (TJP1-α-) is predominantly expressed in tumor tissues and in A549 cells during transforming growth factor-β (TGF-β)-induced EMT. RBM47 promoted the inclusion of exon 20 of TJP1, the alternative exon encoding the α-domain, by which RBM47 recognizes to (U)GCAUG in the downstream intronic region of exon 20. We also found that the first RNA recognition motif (RRM) domain of RBM47 is critical in the regulation of alternative splicing and its recognition to pre-mRNA of TJP1. Furthermore, we demonstrated that the TJP1-α- isoform enhances the assembly of actin stress fibers, thereby promoting cellular migration in a wound healing assay. Our results suggest the regulatory mechanism for the alternative splicing of TJP1 pre-mRNA by RBM47 during EMT, providing a basis for studies related to the modulation of EMT via alternative splicing.

In this paper, we aimed to develop a pulsed electromagnetic field (PEMF) system and to investigate its effects on diverse cell types. To ensure uniform magnetic flux density in the space for cell culture, the PEMF system was designed with an efficient solenoid using an analytical design and finite-element analysis. We investigated its effects on diverse cell types by using cell proliferation assay and flow cytometry analysis. The results of this paper show promise in furthering the PEMF-related biomedical research and its applications in translational studies.

The RNA-binding protein Rbfox3 is a well-known splicing regulator that is used as a marker for post-mitotic neurons in various vertebrate species. Although recent studies indicate a variable expression of Rbfox3 in non-neuronal tissues, including lung tissue, its cellular function in lung cancer remains largely unknown. Here, we report that the number of RBFOX3-positive cells in tumorous lung tissue is lower than that in normal lung tissue. As the transforming growth factor-β (TGF-β) signaling pathway is important in cancer progression, we investigated its role in RBFOX3 expression in A549 lung adenocarcinoma cells. TGF-β1 treatment inhibited RBFOX3 expression at the transcriptional level. Further, RBFOX3 depletion led to a change in the expression levels of a subset of proteins related to epithelial-mesenchymal transition (EMT), such as E-cadherin and Claudin-1, during TGF-β1-induced EMT. In immunofluorescence microscopic analysis, mesenchymal morphology was more prominent in RBFOX3-depleted cells than in control cells. These findings show that TGF-β-induced RBFOX3 inhibition plays an important role in EMT and propose a novel role for RBFOX3 in cancer progression.

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β-tubulin and β-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

Rbfox3, an RNA-binding fox protein, binds to the antibody to pan-neuronal marker, neuronal nuclei (NeuN). Rbfox3 is expressed in neural tissues across a wide range of species including mammals, birds, and amphibians. However, the molecular identity of Rbfox3 in the zebrafish is largely unknown. In this study, we cloned two zebrafish Rbfox3 genes, Rbfox3a and Rbfox3b. We also cloned the Rbfox3-d31 isoform, which excludes a 93-nucleotide alternative exon within the RNA-recognition motif in both, Rbfox3a and Rbfox3b. Multiple protein sequence alignment revealed that the amino acid sequence for residues 1-20 of the zebrafish Rbfox3, which is the epitope region of NeuN antibody, was different from that of other species. Therefore, NeuN antibody lost its function as a neuronal marker antibody in zebrafish. Reverse transcriptase-polymerase chain reaction showed that both Rbfox3-d31 transcripts were abundant in the early blastula stage, after which they dramatically reduced, suggesting that these isoforms exist mainly as maternal transcripts. In contrast, full-length Rbfox3 transcripts were detected from the 24 h post-fertilization embryo, expression was also maintained at a constant level. Furthermore, full-length Rbfox3-expressing cells were located within the central nervous system during later stages of the zebrafish embryo. Our study provides insight into the molecular structure of zebrafish Rbfox3 as a step towards genetic association studies investigating the developmental role of Rbfox3.

Tuberculosis (TB) remains a global healthcare issue. Understanding the host-pathogen interactions in TB is vital to develop strategies and therapeutic tools for the control of Mycobacterium tuberculosis (Mtb). In this study, transcriptome analyses of macrophages infected with either the virulent Mtb strain H37Rv (Rv) or the avirulent Mtb strain H37Ra (Ra) were carried out and 750 differentially expressed genes (DEGs) were identified. As expected, the DEGs were mainly involved in the induction of innate immune responses against mycobacterial infections. Among the DEGs, solute carrier family 7 member 2 (Slc7a2) was more strongly expressed in Ra-infected macrophages. Induction of SLC7A2 was important for macrophages to control the intracellular survival of Mtb. Our results imply that SLC7A2 plays an important role in macrophages during Mtb infection. Our findings could prove useful for the development of new therapeutic strategies to control TB infection.

Clinical application of Ag-specific T regulatory cells (Tregs) offers promise for the treatment of undesirable immune diseases. To achieve this goal, long-term expansion of Tregs is required to obtain sufficient numbers of cells. However, human Tregs are not stable ex vivo. Therefore, we previously developed an innovative Treg expansion protocol using 25mer-phosphorothioated random oligonucleotides (ODNps25). The addition of ODNps25 successfully resulted in the stabilization of engineered Ag-specific Tregs; however, the mechanism is not fully characterized. We first identified sterile α motif histidine-aspartate–domain containing protein 1 (SAMHD1) as an ODNps25-binding protein using a UV–cross-linking pull-down strategy. SAMHD1 physically interacted with the 3′ untranslated region of Foxp3 mRNA and was translocated from nucleus to cytoplasm after ODNps25 treatment. Importantly, addition of ODNps25 enhanced the interaction of SAMHD1 and Foxp3 mRNA significantly, and this interaction was increased by TCR stimulation. Because ODNps25 binds to the nuclease (HD) domain of SAMHD1, we then established that overexpression of a dNTPase-deficient mutant (D137N) in Tregs significantly stabilized the expression level of the Foxp3 protein. Furthermore, we found that TCR stimulation upregulates phosphorylation of the threonine residue (Thr592), which is a regulatory site to control SAMHD1 activity, and phosphorylation of Thr592 is critical to control SAMHD1 activity to stabilize the expression of Foxp3 and Helios in Tregs. Taken together, we suggest that the interaction of ODNPs25 in HD or phosphorylation of Thr592 by TCR stimulation interferes with nuclease activity of SAMHD1, thereby stabilizing 3′ untranslated region of Foxp3 and Helios mRNAs in long-term culture.

Background Calcific tendinopathy (CT) is characterized by deposits of calcium, most commonly found in the shoulder tendons. The exact cause and pathogenesis of CT are not fully understood. This study analyzed the expression pattern of RNA-binding protein fox-1 homolog 2 (RBFOX2), a crucial splicing regulator in tissue differentiation. Methods Normal and calcific tendons were compared for RBFOX2 mRNA level using quantitative reverse-transcription polymerase chain reaction. Intracellular localization of RBFOX2 protein was investigated using immunofluorescence microscopy. Normal and calcific tendon cDNAs were used to clone RBFOX2. Sequencing analysis identified coding sequences of the RBFOX2 isoform. Results The intracellular localization of RBFOX2 protein differed with disease status, with RBFOX2 localized in the cytoplasm in calcific tendons and the nucleus in normal tendons. Analysis of the RBFOX2 protein-coding sequence showed that exon 10, responsible for nuclear localization, was absent in calcific tendons. Splicing of RBFOX2 target genes CHD2 and MBNL1 was significantly affected by cytoplasmic localization of RBFOX2 in calcific tendons. Discussion Given the function of RBFOX2 as a splicing regulator in the nucleus, cytoplasmic localization of RBFOX2 protein in calcific tendons may have affected overall splicing events and altered gene expression. These results provide insights for comprehension of CT pathogenesis.

Rbfox RNA-binding proteins play important roles in the regulation of alternative pre-mRNA splicing, but their role in other gene regulatory mechanisms is not well understood. Here, we show that Rbfox2 is a novel constituent of cytoplasmic stress granules, the translational silencing machinery assembled in response to cellular stress. We also show that the RNA binding activity of the Rbfox family protein is crucial for its localization into stress granules. To investigate the role of Rbfox2 in stress granules we used RNA-immunoprecipitation sequencing to identify cytoplasmic transcriptome-wide targets of Rbfox2. We report that a subset of cell cycle-related genes including retinoblastoma 1 is the target of Rbfox2 in cytoplasmic stress granules, and Rbfox2 regulates the retinoblastoma 1 mRNA and protein expression levels during and following stress exposure. Our study proposes a novel function for Rbfox2 in cytoplasmic stress granules.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Many studies investigating AD pathogenesis and its therapy have been conducted but none have been successful. One of the causes of AD is dysfunction of tight junctions through reduction of claudin 1 expression in the epidermal barrier of the skin. In the present study, we investigated the role of bortezomib (BTZ) in the restoration of the reduced expression of claudin 1. Immunoblot and immunofluorescence analyses revealed that BTZ increased the protein expression level of claudin 1 in the human keratinocyte cell line HaCaT, thereby forming paracellular barriers. Furthermore, repeated application of BTZ alleviated atopic symptoms on the backs and ears of 2, 4-dinitrochlorobenzene (DNCB)-induced AD mice, and led to the formation of normal tight junctions in the epidermal barrier of DNCB-induced mice skin. Taken together, these results demonstrate that BTZ-induced claudin 1 expression may be a valuable therapeutic approach for AD.

Phosphodiesterase 3A (PDE3A), a member of the cGMP-inhibited cyclic nucleotide phosphodiesterase (PDE) family, plays important roles in oocyte maturation and vascular smooth muscle cell proliferation. However, the molecular mechanisms that regulate PDE3A gene expression remain largely unknown. In this study, we investigated the transcriptional regulation of PDE3A , and found that the splicing factor proline and glutamine rich (SFPQ) protein modulated PDE3A mRNA levels. Multiple transcription start sites (TSS1, 2, and 3) were identified within the first exon of PDE3A using 5'-rapid amplification of cDNA ends (RACE). Variable expression levels of three PDE3A variants were also observed in human tissues and HeLa cells. Several putative SFPQ-binding sites were identified upstream of the regulatory region of PDE3A -TSSs using chromatin immunoprecipitation sequencing (ChIP-seq). Serum-induced PDE3A expression was affected by increasing the amount of SFPQ binding to the upstream regulatory region of PDE3A In addition, transcription of PDE3A was lower in human cervical adenocarcinoma cells compared to normal cervical tissue. Furthermore, over-expression of PDE3A induced sensitivity to anti-cancer therapeutic agent, 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), in HeLa cells. Taken together, these results suggest that SFPQ functions as a transcriptional activator of PDE3A, which is involved in the regulation of DNMDP sensitivity , offering a novel molecular target for the development of anticancer therapies.

Magnesium (Mg) ions are the most abundant intracellular divalent cations and play a pivotal role in numerous cellular processes. Biodegradable Mg-containing materials, including scaffolds, are promising candidates for orthopedic applications. Here, we investigated the effect of Mg ions on the cellular properties of osteoblasts. Cytotoxicity tests on osteoblasts confirmed that no cytotoxic effects were found up to a supplementing Mg ion concentration of 10 mM. Mg ions at a concentration of 5 mM increased the migration and invasiveness of osteoblasts. To investigate the stimulatory effect of Mg ions on cell motility in scaffolds, we fabricated 10 wt% Mg ion-containing polycaprolactone (PCL) scaffolds, using the wire-network molding (WNM) technique. Mg ion-containing scaffolds persistently released Mg ions at a concentration of 5 mM in the media after pre-incubation. Furthermore, increased cell motility was confirmed in Mg ion-containing scaffolds by quantification of genomic DNA and protein content. Our results provide an important basis for the function of Mg ions and their effect on cell motility, and propose a novel role for Mg ions in scaffold applications.

RBFOX3, a nuclear RNA-binding protein, is well known as a regulator of alternative pre-mRNA splicing during neuronal development. However, other functions of RBFOX3 are poorly understood. Here, we investigated the function of RBFOX3 in the cytoplasm with respect to regulation of Claudin-1 expression. In human lung tissue, Claudin-1 is higher in RBFOX3-positive cells than in RBFOX3-negative cells. Immunostaining and mRNA quantification revealed that protein levels, but not mRNA levels, of Claudin-1 are increased by RBFOX3. In addition, cycloheximide treatment of human lung cancer cells revealed that RBFOX3 increases the stability of Claudin-1 through attenuation of its ubiquitination. Our study provides insights into the molecular mechanisms by which RBFOX3 regulates Claudin-1 expression in human lung tissue.

RNA-binding proteins (RBPs) regulate numerous aspects of gene expression; thus, identification of their endogenous targets is important for understanding their cellular functions. Here we identified transcriptome-wide targets of Rbfox3 in neuronally differentiated P19 cells and mouse brain by using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP). Although Rbfox3 is known to regulate pre-mRNA splicing through binding the UGCAUG motif, PAR-CLIP analysis revealed diverse Rbfox3 targets including primary microRNAs (pri-miRNAs) that lack the UGCAUG motif. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs. In vitro analyses revealed that Rbfox3 functions as a positive and a negative regulator at the stage of pri-miRNA processing to precursor miRNA (pre-miRNA). Rbfox3 binds directly to pri-miRNAs and regulates the recruitment of the microprocessor complex to pri-miRNAs. Our study proposes a new function for Rbfox3 in miRNA biogenesis.

Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin-proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo.

Rbfox3 is required to promote neuronal differentiation of postmitotic neurons through Numb alternative splicing., Alternative premRNA splicing is a major mechanism to generate diversity of gene products. However, the biological roles of alternative splicing during development remain elusive. Here, we focus on a neuron-specific RNA-binding protein, Rbfox3, recently identified as the antigen of the widely used anti-NeuN antibody. siRNA-mediated loss-of-function studies using the developing chicken spinal cord revealed that Rbfox3 is required to promote neuronal differentiation of postmitotic neurons. Numb premRNA encoding a signaling adaptor protein was found to be a target of Rbfox3 action, and Rbfox3 repressed the inclusion of an alternative exon via binding to the conserved UGCAUG element in the upstream intron. Depleting a specific Numb splice isoform reproduced similar neuronal differentiation defects. Forced expression of the relevant Numb splice isoform was sufficient to rescue, in an isoform-specific manner, postmitotic neurons from defects in differentiation caused by Rbfox3 depletion. Thus, Rbfox3-dependent Numb alternative splicing plays an important role in the progression of neuronal differentiation during vertebrate development.

Growth arrest and DNA damage-inducible beta (GADD45b) plays a pivotal role in many intracellular events in both cell survival- and cell death-related signaling. To date, the study of GADD35b has mainly focused on investigation of its function, as well as interacting molecules. However, studies of Gadd45b gene regulation are limited. In this study, we investigated the transcriptional regulation mechanism of Gadd45b. Since Gadd45b mRNA is highly induced by the PPARα agonist Wy-14,643 in the mouse liver, we analyzed the Gadd45b promoter using an in vivo reporter assay. Interestingly, the naked Gadd45b-luciferase construct strongly induced luciferase activity without any stimulant in our in vivo system. Therefore, we investigated the epigenetic changes in the Gadd45b promoter region using mouse liver genomic DNA, the methylation-specific restriction enzyme (HpaII), and disulfide conversion. Our results showed that two possible CpG methylation sites were methylated and demethylated by Wy-14,643 treatment. This study indicates that epigenetic change at the Gadd45b promoter is critical for Gadd45b induction.

Although the regulation of thyroid stimulating hormone β-subunit gene (TSHβ) has been intensively studied, the functions of transcription factors involved are not fully understood. The authors found that the −615/−516 promoter region of the TSHβ interacts specifically with nuclear proteins derived from pituitary tissue or from cultured thyrotroph cells. The actual binding site at the nucleotide level, as revealed by DNase I protection assay, includes the consensus sequence for nuclear factor I (NFI). RT-PCR analysis indicated that NFI-B expression is restricted to thyrotroph cells in the anterior pituitary. EMSA and ChIP analysis showed that NFI-B binds most efficiently to the −588/−560 region of TSHβ promoter. The forced expressions of NFI-B markedly reduced TSHβ promoter activity and its mRNA expression. Furthermore, it was also shown that the −588/−560 region is involved in the insulin-mediated repression of the TSHβ. It was of particular interest to observe that NFI-B was recruited to the −588/−560 region of the TSHβ promoter in an insulin-dependent manner. Taken together, this study provides new insights of the delicate regulations of energy metabolism and hormonal homeostasis.

Statins are widely used drugs for the treatment of hypercholesterolaemia. A number of recent studies have suggested that statins also have pleiotropic effects on immune responses and statins have proven to be effective in the treatment of autoimmune diseases in animal models. Foxp3(+) T regulatory cells are a unique subset of CD4(+) T cells that mediate immunosuppression. Foxp3(+) T cells develop in the thymus, but can also be induced in peripheral sites in the presence of transforming growth factor-beta (TGF-beta). We demonstrate here that simvastatin blockade of the mevalonate pathway can mediate induction of mouse Foxp3(+) T cells and that simvastatin can synergize with low levels of TGF-beta to induce Foxp3(+) T cells. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated at late time-points after T-cell activation, and are associated with demethylation of the Foxp3 promoter. One major effect of simvastatin was inhibition of the induction of Smad6 and Smad7, inhibitory Smads that inhibit TGF-beta signalling. Our results suggest that one mechanism responsible for the immunosuppressive effects of statins is the ability to promote the generation of Foxp3(+) T regulatory cells.

To clarify the regulatory mechanism of GW112 gene expression, 5'-flanking region of the human GW112 gene was isolated and characterized in the present study. 5'-RACE analysis showed a single transcription start site, which is located 142 nucleotides upstream of the translation initiation site. Transient transfection studies with serial deletion constructs and close examination of the sequences identified a putative NF kappaB binding sequence between -442 and -430, which could be responsible for efficient expression of the GW112 gene. Indeed, GW112 gene was found to be regulated by NF kappaB signals including overexpressed p65 and I kappaB alpha, IKK inhibitor, and proteasome inhibitor. Binding of NF kappaB to its putative site was confirmed by EMSA and ChIP assays. These results suggest that NF kappaB is an essential regulatory factor for GW112 transcription. Based on this finding, we next confirmed that inhibition of GW112 expression could induce apoptosis in the presence of cytotoxic agent in gastric cancer cells. Furthermore, knocking-down or overexpression of GW112 gene in gastric cancer cells demonstrated that GW112 has an antiapoptotic property against the cytotoxic agents-induced apoptosis. Taken together, these results suggest that GW112 could be an important mediator in NF kappaB-dependent tumorigenesis of digestive tract tissues.

Studying the regulatory mechanism of the glycoprotein hormone α subunit (αGSU) gene in thyrotropes is essential for understanding the synthesis of functional thyroid-stimulating hormone (TSH). Here, we investigated the influence of a homeodomain transcription factor Msx1 (Msh homeobox 1) on αGSU expression in thyrotropes. The transient expression of Msx1 inhibited the activity of an αGSU reporter gene, as well as its endogenous mRNA level in thyrotrope-derived αTSH cells. Luciferase reporter assays with serial deletion constructs and a close examination of the sequences revealed that the putative Msx1 binding site (PMS) in the αGSU promoter is not responsible for Msx1-mediated transcriptional repression. We also identified the TATA-box binding protein (TBP) as an interacting protein in thyrotropes. Interaction of TBP with Msx1 attenuates the inhibitory effect of Msx1 on αGSU gene expression in a DNA binding-independent manner. Furthermore, transient transfection studies with mutant Msx1 revealed that the interaction of TBP and Msx1 is critical for Msx1-mediated transcriptional repression of the αGSU. These results suggest that Msx1 functions as a transcriptional repressor of αGSU and that its interaction with TBP is an integral part of the mechanism by which Msx1 regulates the inhibition of αGSU gene expression.

1-Alkoxy-5-alkyluracils 2a-f have been prepared by the reaction of 2-alkyl-3-methoxyacryloyl isocyanates 8a-b with alkoxyamines 9a-c followed by cyclization of the resulting N-alkoxy-N'-(2-alkyl-3-methoxyacryloyl)ureas 10a-f. The isocyanates 8a-b were prepared from ethyl 2-alkylacrylates 3a-b in 5 steps.

Olfactomedin 4 (OLFM4) is highly expressed in gastrointestinal cancers and has an anti-apoptotic function. The roles of OLFM4 in tumor growth and metastasis and how it functions in these processes remain elusive. We investigated the function of OLFM4 in tumor growth and metastasis using B16F10 mouse melanoma cells as an experimental system. Our results showed that OLFM4 had no positive effect on cell viability or cell cycle progression in B16F10 cells. However, it significantly suppressed the tumorigenicity of B16F10 cells, i.e., intradermal primary tumor growth and lung metastasis. OLFM4 also suppressed the migration and invasion of B16F10 cells in vitro. For further insight into the mechanisms underlying OLFM4-mediated suppression of tumor progression, we examined the effect of OLFM4 on the expression of integrin and matrix metalloproteinase (MMP), both of which are involved in tumor progression. Overexpression of OLFM4 clearly reduced the expression levels of integrin α1, integrin α4, integrin α5, integrin α6, and MMP9. Moreover, forced expression of MMP9 attenuated the inhibitory activity of OLFM4 on migration and invasiveness. Our findings provide the experimental evidence that OLFM4 may function as a tumor suppressor and an anti-metastatic gene during tumor progression.

Lhx2, a member of LIM homeobox transcription factors, plays a key role in central nervous system (CNS) and embryonic tissue development. However, molecular mechanism of Lhx2 gene regulation remains largely unknown. Here, we identified and characterized a regulatory region of Lhx2 gene which mediates responses to two different signals such as inhibition of HDAC3 and stimulation by E2F1. In particular, the promoter region of −229 to −126 was responsible not only for basal expression but also for a inhibitor of histone deacetylase, trichostatin A (TSA)-mediated activation of Lhx2 gene. Intriguingly, transcription factor E2F1 also activates Lhx2 gene via direct binding to the same −229 to −126 region. Based on these observations, we could have demonstrated that E2F1 is necessary for TSA-mediated activation of Lhx2 gene and acetylation of histone 3 is involved in this event. This study provides evidence that the histone modification and E2F1 binding are integral parts of the mechanism for Lhx2 gene expression.

Fox-1 family (Fox) proteins, which consist of Fox-1 (A2BP1), Fox-2 (Rbm9) and Fox-3 (NeuN) in mammals, bind to the RNA element UGCAUG and regulate alternative pre-mRNA splicing. However the mechanisms for Fox-regulated splicing are largely unknown. We analyzed the expression pattern of the three Fox proteins as well as neural cell-specific alternative splicing of a cassette exon N30 of nonmuscle myosin heavy chain (NMHC) II-B in the mouse central nervous system. Histological and biochemical analyses following fluorescence-activated cell sorting demonstrate a positive correlation of N30 inclusion and Fox-3 expression. Further, we identified polypyrimidine tract binding protein-associated splicing factor (PSF) as an interacting protein with Fox-3 by affinity-chromatography. In cultured cells, enhancement of N30 inclusion by Fox-3 depends on the presence of PSF. PSF enhances N30 inclusion in a UGCAUG-dependent manner, although it does not bind directly to this element. Fox-3 is recruited to the UGCAUG element downstream of N30 in the endogenous NMHC II-B transcript in a PSF-dependent manner. This study is the first to identify PSF as a coactivator of Fox proteins and provides evidence that the Fox-3 and PSF interaction is an integral part of the mechanism by which Fox proteins regulate activation of alternative exons via a downstream intronic enhancer.

1-Alkoxy-5-alkyluracils 2a-f have been prepared by the reaction of 2-alkyl-3-methoxyacryloyl isocyanates 8a-b with alkoxyamines 9a-c followed by cyclization of the resulting N-alkoxy-N'-(2-alkyl-3-methoxyacryloyl)ureas 10a-f. The isocyanates 8a-b were prepared from ethyl 2-alkylacrylates 3a-b in 5 steps.

To clarify the regulatory mechanism of GW112 gene expression, 5'-flanking region of the human GW112 gene was isolated and characterized in the present study. 5'-RACE analysis showed a single transcription start site, which is located 142 nucleotides upstream of the translation initiation site. Transient transfection studies with serial deletion constructs and close examination of the sequences identified a putative NF kappaB binding sequence between -442 and -430, which could be responsible for efficient expression of the GW112 gene. Indeed, GW112 gene was found to be regulated by NF kappaB signals including overexpressed p65 and I kappaB alpha, IKK inhibitor, and proteasome inhibitor. Binding of NF kappaB to its putative site was confirmed by EMSA and ChIP assays. These results suggest that NF kappaB is an essential regulatory factor for GW112 transcription. Based on this finding, we next confirmed that inhibition of GW112 expression could induce apoptosis in the presence of cytotoxic agent in gastric cancer cells. Furthermore, knocking-down or overexpression of GW112 gene in gastric cancer cells demonstrated that GW112 has an antiapoptotic property against the cytotoxic agents-induced apoptosis. Taken together, these results suggest that GW112 could be an important mediator in NF kappaB-dependent tumorigenesis of digestive tract tissues.

NeuN (neuronal nuclei) is a neuron-specific nuclear protein which is identified by immunoreactivity with a monoclonal antibody, anti-NeuN. Anti-NeuN has been used widely as a reliable tool to detect most postmitotic neuronal cell types in neuroscience, developmental biology, and stem cell research fields as well as diagnostic histopathology. To date, however, the identity of its antigen, NeuN itself, has been unknown. Here, we identify NeuN as the Fox-3 gene product by providing the following evidence: 1) Mass spectrometry analysis of anti-NeuN immunoreactive protein yields the Fox-3 amino acid sequence. 2) Recombinant Fox-3 is recognized by anti-NeuN. 3) Short hairpin RNAs targeting Fox-3 mRNA down-regulate NeuN expression. 4) Fox-3 expression is restricted to neural tissues. 5) Anti-Fox-3 immunostaining and anti-NeuN immunostaining overlap completely in neuronal nuclei. We also show that a protein cross-reactive with anti-NeuN is the synaptic vesicle protein, synapsin I. Anti-NeuN recognizes synapsin I in immunoblots with one order of magnitude lower affinity than Fox-3, and does not recognize synapsin I using immunohistology. Fox-3 (also called hexaribonucleotide-binding protein 3 and D11Bwg0517e) contains an RNA recognition motif and is classified as a member of the Fox-1 gene family that binds specifically to an RNA element, UGCAUG. We demonstrate that Fox-3 functions as a splicing regulator using neural cell-specific alternative splicing of the non-muscle myosin heavy chain II-B pre-mRNA as a model. Identification of NeuN as Fox-3 clarifies an important element of neurobiology research.

Although there is evidence that the LIM homeodomain transcription factor, Lhx2, can stimulate transcription of the glycoprotein hormone alpha-subunit gene, the role of Lhx2 in regulating TSH beta-subunit has not been established. In the present studies, the ability of Lhx2 to regulate transcription of the TSH beta-subunit gene was examined. In the thyrotrope-derived TalphaT1 cell line, Lhx2 expression was found to be induced by treatment with either TRH or cAMP, consistent with the possibility that Lhx2 may play a role in mediating the ability of this signaling pathway to stimulate TSH gene expression. Transient, forced overexpression of Lhx2 stimulated activity of a TSH beta-subunit reporter gene. Deletion studies provided evidence that the -177 to -79 region of the TSH beta-subunit promoter was necessary for stimulation of reporter gene activity by Lhx2. A gel mobility shift assay provided the evidence that Lhx2 can bind to this region of DNA. DNase I footprinting studies demonstrated that two distinct regions of the TSHbeta promoter, -118 to -108 and -86 to -68, are protected by Lhx2 from nuclease digestion. These regions contain repeats of the sequence, 5'-(G/T)CAAT(T/A)-3'. Mutation of this sequence, especially in the -86 to -68 region, substantially decreased Lhx2 responsiveness of the TSH beta-subunit reporter gene. In addition, a DNA fragment containing the -177 to -79 region of the TSHbeta promoter was found to confer Lhx2 responsiveness to a minimal promoter. These results provide multiple lines of evidence consistent with a role for Lhx2 in modulating expression of the TSH beta-subunit gene.

Various salivary glands in senescent humans and other animals have been examined extensively to characterize the structural and functional changes that occur during aging. Although a wide range of different structural changes, involving both the parenchymal and stromal tissues, have been described, it is unclear how any of these changes affects the function of the salivary glands. One major change in structure is the reduction in the volume of acini with a concomitant increase in the ductal volume. Despite this loss of functional acini, the salivary output and the contents seem to be unaltered, or minimally altered, due to aging. One consistent change observed in many salivary glands of aged animals is the decline in the rate of synthesis of proteins and their messenger RNA (mRNA). However, the salivary acinar cells from aged animals can synthesize secretory proteins at an elevated rate just as effectively as those from their younger counterparts in response to external stimuli, which are known to enhance the rate of protein synthesis. Thus, it appears that the salivary acinar cells, which remain structurally intact during aging, seem to retain their functional efficiency. Furthermore, these acinar cells, although reduced in number, are sufficient in quantity to carry out most of the salivary gland functions.