Identification of transcriptional regulatory elements in the human somatostatin receptor sst2 promoter and regions including estrogen response element half-site for estrogen activation.

The somatostatin receptor subtype 2 (sst2) mediates inhibition of hormone secretion and cell proliferation, and modulates neurotransmission. Its expression is widespread in various normal tissues and many malignant cells, and is up-regulated by estrogen in breast cancer cells. This study was undertaken to investigate molecular mechanism of transcriptional regulation of the human sst2 gene, for which an additional exon (exon 1) in the 5'-untranslated region was recently found. Transient transfection and mutational analysis showed that the immediate 5'-upstream region containing two Sp1 (-54/-45 and -88/-79) and an ATF/CRE (-69/-62) sites provided full promoter activity. An EMSA together with transfection analysis in Sp1-deficient Drosophila Schneider line (SL2) cells showed that Sp1 acted on the proximal Sp1 site, whereas Sp3, Sp1, and Sp2 did on the distal Sp1 site. Activating transcription factor-2 (ATF)-2, c-Jun, and cyclic AMP response element-binding protein (CREB) interacted with the ATF/CRE site. Transcriptional activation by estrogen occurred through two different regions; one included these proximal elements and the other existed in the upstream region containing estrogen response element (ERE) half-site (-348/-344) and GC-rich sequence (-447/-414). This upstream estrogen responsiveness was observed in a human breast cancer T47D cell, but not in GH(3) or estrogen receptor alpha (ERalpha) -expressing HeLa cells, and was potentiated by overexpression of ERalpha or ERbeta, whose binding to the ERE half-site was verified by EMSAs. A chromatin immunoprecipitation assay suggested that ERalpha was recruited to the ERE half-site after estrogen treatment in T47D cells. The present results should provide a molecular basis for transcriptional regulation in a variety of physiological and pathological contexts of sst2-expressing tissues.

Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease.

Citrullinated proteins are the products of a posttranslational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion-dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimer's disease (AD). By Western blot analysis with antimodified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, but the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and the normal hippocampal areas. PAD2 enrichment coincided well with citrullinated protein positivity. Double immunofluorescence staining revealed that citrullinated protein- and PAD2-positive cells also coincided with GFAP-positive cells, but not all GFAP-positive cells were positive for PAD2. As with GFAP, which is an astrocyte-specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may become a useful marker for human neurodegenerative diseases.

Proteomics-based identification of differentially expressed genes in human gliomas: down-regulation of SIRT2 gene.

A number of chromosomal abnormalities including 19q deletions have been associated with the formation of human gliomas. In this study, we employed a proteomics-based approach to identify possible genes involved in glioma tumorigenesis which may serve as potential diagnostic molecular markers for this type of cancer. By comparing protein spots from gliomas and non-tumor tissues using two-dimensional (2D) gel electrophoresis, we identified 11 up-regulated proteins and four down-regulated proteins in gliomas. Interestingly, we also discovered that a group of cytoskeleton-related proteins are differentially regulated in gliomas, suggesting the involvement of cytoskeleton modulation in glioma pathogenesis. We then focused on the cytoskeleton-related protein, SIRT2 (sirtuin homologue 2) tubulin deacetylase, which was down-regulated in gliomas. SIRT2 is located at 19q13.2, a region known to be frequently deleted in human gliomas. Subsequent Northern blot analysis revealed that RNA expression of SIRT2 was dramatically diminished in 12 out of 17 gliomas and glioma cell lines, in agreement with proteomic data. Furthermore, ectopic expression of SIRT2 in glioma cell lines led to the perturbation of the microtubule network and caused a remarkable reduction in the number of stable clones expressing SIRT2 as compared to that of a control vector in colony formation assays. These results suggest that SIRT2 may act as a tumor suppressor gene in human gliomas possibly through the regulation of microtubule network and may serve as a novel molecular marker for gliomas. Additional proteins were also identified, whose function in gliomas was previously unsuspected.

Molecular evolution of nucleoside diphosphate kinase genes: conserved core structures and multiple-layered regulatory regions.

Genomic data regarding the nucleoside diphosphate (NDP) kinase genes have been accumulated from diverged phyla. Comparison of their regulatory sequences have shed light on the multiple facets of gene regulation systems. Phylogenetic studies, including CpG island and intron-mapping, and homologous sequence comparison, have suggested that the regions of the major mammalian genes, the ortholog (rat alpha or nm23-H2) and its paralog (rat beta or nm23-H1), have been constructed by a stepwise gain and loss of alien genes resulting in "multiple-layered" regulatory systems. They contain representative cis-elements for the constitutive, stage/lineage-specific, and early response expression. These elements' binding capacities to nuclear proteins were confirmed by electrophoretic mobility shift assay. Further, these regulatory systems generate heterogeneous mRNA at the 5' untranslated region, which influences their own translation efficiencies. In terms of this process, the transcription system would control another layer of gene expression: posttranscriptional (translational) regulation.

Nucleoside diphosphate kinases in mammalian signal transduction systems: recent development and perspective.

The role of nucleoside diphosphate (NDP) kinase with special reference to mammalian signal transduction systems was described. The interaction between NDP kinases and G proteins was reevaluated in view of their protein structural information and its significance was extended further on the basis of recent findings obtained with small molecular weight G proteins such as Rad, menin, and Rac. Meanwhile, observations suggesting involvement of NDP kinases in the regulation of cell growth and differentiation led to the realization that NDP kinases may play a crucial role in receptor tyrosine kinase signal transduction systems. In fact, a number of experimental results, particularly obtained with PC12 cells, implicate that NDP kinases appear to regulate differentiation marker proteins and cell-cycle-associated proteins cooperatively. Consequently, we propose a hypothesis that NDP kinases might act like a molecular switch to determine the cell fate toward proliferation or differentiation in response to environmental signals.