OBJECTIVETo characterize the deficiency of the mRNA expression of specific protein (SP3) gene in peripheral blood mononuclear cells (PBMCs) from Chinese patients with multiple sclerosis (MS) and study its correlation with the disease phenotypes.

METHODSFifty-six patients with definite MS were collected and total RNA was extracted from their PBMCs. Specific primers corresponding to SP3 gene were designed and the mRNA expression of SP3 gene was detected by reverse transcriptase-PCR (RT-PCR) method. The deficiency of SP3 expression was compared among MS patients, irrelevant disease group and normal controls.

RESULTSOf the 56 MS cases, 23 (41.1%) were SP3-deficient. In contrast, the frequency of SP3-deficiency in normal subjects and irrelevant disease controls was 8.6% (5/35) and 14.3% (4/27), respectively. The frequency of the SP3-expression deficiency in MS patients was significantly higher than that in both control groups (P< 0.01). Within the MS cases, the scores of expanded disability status scale (EDSS) in the SP3-expressing subjects were significantly different from that in the SP3-deficient ones in the stable, but not in the active, phase of MS (P< 0.05).

CONCLUSIONAuthor's observation suggested that deficient expression of SP3 gene occurs in Chinese MS patients, and that the SP3 expression may correlate with the clinical manifestations of MS and play roles in its immunological pathogenesis.

Adolescent ; Adult ; Aged ; Child ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Multiple Sclerosis ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sp3 Transcription Factor ; genetics ; Young Adult

We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.
Cell Line, Tumor ; Electrophoretic Mobility Shift Assay ; Histone Deacetylases/*antagonists & inhibitors/metabolism ; Humans ; Hydroxamic Acids/*pharmacology ; Promoter Regions, Genetic ; Sp1 Transcription Factor/genetics/*metabolism ; Sp3 Transcription Factor/genetics/metabolism ; Stomach Neoplasms/*metabolism ; Transcription, Genetic ; Tumor Suppressor Proteins/*biosynthesis/genetics ; p300-CBP Transcription Factors/genetics/metabolism

OBJECTIVETo investigate the role of Sp3 in the transcriptional regulation of enamelin gene.

METHODSBy bioinformatic analysis, a putative responsive element for Sp3 was identified. Electrophoretic mobility shift assay was used to examine the interaction between Sp3 and enamelin. 5'-flanking regulatory region of enamelin was cloned and ligated into pGL3-basic luciferase vector. Sp3 and the Enam-luc were cotransfected into mouse ameloblast-like cell line, and the activity of luciferase was examined.

RESULTSThe results showed that Sp3 could not directly bind to the enamelin regulation region and activate enamelin transcription.

CONCLUSIONSSp3 might not be involved in transcriptional regulation of enamelin gene via an indirect interaction.

5' Flanking Region ; genetics ; Ameloblasts ; cytology ; Animals ; Cell Line ; Dental Enamel Proteins ; genetics ; metabolism ; Electrophoretic Mobility Shift Assay ; Female ; Gene Expression Regulation ; Genes, Reporter ; Luciferases ; Male ; Mice ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Regulatory Elements, Transcriptional ; Sp3 Transcription Factor ; genetics ; metabolism ; Transcription, Genetic ; Transcriptional Activation ; Transfection

PURPOSE: FK506 (tacrolimus) is a widely used immunosuppressive agent in the treatment of various medical conditions, including autoimmune disease, bone marrow and organ transplantations. Previously FK506 was known to cause apoptotic death of human Jurkat T cells. METHODS: The current study was designed to analyze the gene expression pattern of Jurkat T cells after FK506 application by using cDNA microarray. Treatment of Jurkat T cells with FK506 resulted in a decrease of cell viability in a time- and dose-dependent manner. Next, total RNA of Jurkat T cells was extracted by using TRIzol reagent and used to carry out a confirmation test for the purity and integrity of total RNA. RESULTS: Gene expression levels related to apoptosis and cell cycle process were mainly focused to analyze in FK506-treated Jurkat T cells. According to the inhibition of calcineurin activity, MARCKS in PKC substrates and Sp3 transcription factor was markedly increased in FK506-treated cells. Also, cell cycle control gene Id1 and Id3 were induced in expression from FK506-treated Jurkat T cells. However, FK506 decreased the expression of Src homology 2, G protein, and MEK 2 genes in bioactive peptide induced signaling pathway. It also reduced the expression level of the insulin receptor, DRPLA and Bai1-associated protein 2 genes, which are involved in the regulation of cell motility and morphology control. CONCLUSION: The author will continue to pursue the exact functional roles of genes that are markedly changed in expression by FK506 in human Jurkat T cells in vitro and in vivo experimental models.
Apoptosis ; Autoimmune Diseases ; Bone Marrow ; Calcineurin ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Movement ; Cell Survival ; Gene Expression ; Gene Expression Profiling ; GTP-Binding Proteins ; Guanidines ; Humans ; Jurkat Cells ; Models, Theoretical ; Oligonucleotide Array Sequence Analysis ; Organ Transplantation ; Phenols ; Receptor, Insulin ; RNA ; Sp3 Transcription Factor ; T-Lymphocytes ; Tacrolimus ; Transplants