Biomedical subjects

Toshitaka Shin

Publications and source records attributed to Toshitaka Shin.

3 recordsLinked to original sources

PubMed2006

Multifactor complex containing B element binding factor, BBF, and repressors regulate the human alpha 1(III) collagen gene (COL3A1).

Type III collagen is found in fetal skin and blood vessels. Previously, we characterized the proximal promoter of the human alpha1(III) collagen gene (COL3A1) using the human rhabdomyosarcoma cell line, A204, and NIH3T3 cells (Yoshino et al., Biochim Biophys Acta, 2005). In the present study, we further analyzed this promoter using additional cell lines, namely a human embryonal rhabdomyosarcoma cell line (RD) and bovine vascular smooth muscle cells (vSMCs), both of which show high expression of type III collagen. Using a luciferase assay, electrophoretic mobility shift assays (EMSA), and DNase footprinting assay, 2 types of multifactor complexes were shown to bind to the DNA region in the vicinity of the B element (- 80 to - 58), depending on the cell type. Next, we used cells stably transfected with a GFP-linked type III collagen promoter fragment for analysis of promoter expression. Usually, transfected cells retained the characteristics of the original cells. However, in several clones derived from RD cells, promoter expression as well as cell shape changed to patterns characteristic of the A204 cell line. Nuclear factors expressed by these clones were also characteristic of the A204 line.

Animals↗

PubMed2005

Multiple proteins are involved in the protein-DNA complex in the proximal promoter of the human alpha1(III) collagen gene (COL3A1).

We have characterized the proximal promoter of the human alpha1(III) collagen gene (COL3A1). Transient transfection assays using a series of chimeric constructs linked to the luciferase gene indicated that the segment from -96 to -34 is necessary to activate transcription. Electrophoretic mobility shift assays (EMSAs) showed that the multiple proteins form the DNA-protein complex in different combinations depending on the cell types. A competition assay using mutant oligonucleotides showed that the sequence 5'-GCTCTCATATTTCAGAA-3' (-79 to -63 bp) is critical for DNA-protein complex formation. This sequence is contained in the B element of mouse alpha1(III) collagen gene (Col3a1) reported by Ruteshouse and de Crombrugghe (J. Biol. Chem., 1993). In the rhabdomyosarcoma cell line, A204, at least two proteins of 92-118 kDa and 40-52 kDa are involved in the DNA-protein complex bound to this motif.

Animals↗

PubMed2005

Sp1 and Sp3 transcription factors upregulate the proximal promoter of the human prostate-specific antigen gene in prostate cancer cells.

The serum level of prostate-specific antigen (PSA) is useful as a clinical marker for diagnosis and assessment of the progression of prostate cancer, and in evaluating the effectiveness of treatment. We characterized four Sp1/Sp3 binding sites in the proximal promoter of the PSA gene. In a luciferase assay, these sites contributed to the basal promoter activity in prostate cancer cells. In an electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we confirmed that Sp1 and Sp3 bind to these sites. Overexpression of wild-type Sp1 and Sp3 further upregulated the promoter activity, whereas overexpression of the Sp1 dominant-negative form or addition of mithramycin A significantly reduced the promoter activity and the endogenous mRNA level of PSA. Among the four binding sites, a GC box located at nucleotides -53 to -48 was especially critical for basal promoter activity. These results indicate that Sp1 and Sp3 are involved in the basal expression of PSA in prostate cancer cells.

Binding Sites↗