The Sp1 and CBF/NF-Y Transcription Factors Cooperatively Regulate the Mouse Pro-alpha3(V) Collagen Gene (Col5a3) in Osteoblastic Cells

The purpose of this study was to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Transient transfection into rat osteosarcoma ROS17/2.8 cells demonstrated that a region from nucleotides 337 to 1 was involved in the transcriptional activity of the Col5a3 gene. An electrophoretic mobility shift assay showed that Sp1/Sp3 and CBF/NF-Y bound to a GC-rich domain (194/186) and a CCAAT box (134/130) in the Col5a3 gene, respectively. Introduction of mutations or deletion into a GC-rich domain, the CCAAT box, or both elements decreased the transcription activity. Overexpression of Sp1 increases the transcription activity and interferes with Sp family binding to the GC-rich domain to decrease promoter activity. Therefore, the transcription of the mouse Col5a3 gene is cooperatively regulated by Sp1 and CBF/NF-Y in osteoblastic cells.</p

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.</p

Esophageal muscle physiology and morphogenesis require assembly of a collagen XIX–rich basement membrane zone

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide–dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, namely impaired smooth-to-skeletal muscle cell conversion in the abdominal segment of the esophagus. This developmental abnormality was accounted for by failed activation of myogenic regulatory factors that normally drive esophageal muscle transdifferentiation. Therefore, these findings identify collagen XIX as the first structural determinant of sphincteric muscle function, and as the first extrinsic factor of skeletal myogenesis in the murine esophagus

Prevention of the initial testosterone surge induced by a luteinizing hormone-releasing hormone analogue in prostate cancer patients:the endocrinological effects of pretreatment with chlormadinone acetate

We investigated the endocrinological effects of pretreatment with chlormadinone acetate (CMA) in preventing the initial testosterone surge induced by a luteinizing hormone-releasing hormone (LH-RH) analogue. A total of 25 patients with previously untreated prostate cancer were included in this study. The patients were randomly assigned to 2 treatment groups:Group1;CMA therapy was begun 4 weeks before the initial LH-RH analogue injection. Group 2;CMA therapy was begun 2 weeks before the initial LH-RH analogue injection. After the initial LH-RH analogue injection, CMA was administered during this study. After LH-RH analogue application, the mean values of serum luteinizing hormone (LH) and testosterone increased in both groups on day 3. However, LH and testosterone levels remained beneath pretreatment values in both groups. The mean relative PSA levels did not significantly increased on day 3 and day 7in both groups. Our results indicate that pretreatment with CMA for 2 weeks was sufficient to prevent the initial testosterone surge in the maximal androgen blockade which was associated with CMA

cDNA Cloning of Human Mitochondrial Transcription Factor 1 with Polymerase Chain Reaction and Overexpression of the Factor in Escherichia coli

A human cDNA encoding mitochondrial transcription factor 1 (mtTF1) was isolated by screening a HeLa cell cDNA library by using polymerase chain reaction. In the present experiment, mtTF1 cDNA clone could be obtained more rapidly, easily and at a low cost as compared with conventional cloning techniques. The size of the cDNA fragent was approximately 2 kb, indicating that the cDNA is almost full length. For the mass production of mtTF1 as a fused protein, the cDNA fragment was inserted into an expression vector pUC19. The recombinant plasmid encodes the mature mtTF1 protein and extra 20 amino acids at its amino terminus derived from Lac Z\u27 in the vector and signal peptide of mtTF1. The fused protein of 26 kDa was overproduced in Escherichia coli, and had the same characteristics of DNA binding as natural mtTF1