Trichostatin A activates the osteopontin gene promoter through AP1 site

In this study, we investigated osteoblastic differentiation by trichostatin A (TSA), a histone deacetylase inhibitor in mouse undifferentiated mesenchymal cell line. TSA increased the osteopontin (OPN) mRNA level and OPN protein. Deletion analysis of the promoter region revealed TSA-induced luciferase response was regulated by -75 to -65 of the OPN promoter. There was an AP1-binding sequence at the site of the OPN promoter. In an electrophoretic mobility shift assay, bands of the complexes were supershifted by addition of antibody to c-fos and phosphorylated c-jun. These data suggested that AP1 plays a crucial role in the TSA-induced OPN expression.     

拟南芥生长素受体基因TIR1启动子的初步分析

以野生型拟南芥（Arabidopsis Columbia）基因组DNA为模板,通过PCR扩增得到拟南芥生长素受体基因T1R1启动子的5个不同长度的系列缺失片段,将这些片断分别克隆到PGM—T载体上。序列分析表明,该启动子系列缺失片段的大小分别为2008bp、1524bp、939bp、532bp和321bp。与已报道的序列完全相同。将不同长度的启动子克隆片断分别与GUS基因融合,构建成表达载体后,在烟草叶片中作遗传转化。分析结果显示：不同长度的启动子片段已整合到烟草基因组中并有GUS酶活性存在,且不同长度启动子片段的表达活性有较明显差异。     

Binding characteristics of a membrane receptor that recognizes 1α25-dihydroxyvitamin D3 and its epimer, 1β,25-dihydroxyvitamin D3

The Steroid hormon 1α, @5-Dihydroxyvitamin D₃ has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast-like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non-genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25-dihydroxyvitamina D₃ can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of ³H-1α,25dihydroxyvitamin D₃ by vitamin D compounds, as well as the apparent dissociation constant of 1α25-dihydroxyvitamin D₃ and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25-dihydroxyvitamin D₃, 7.25 nM to 725 nM, displaced ³H-1α25-dihydrxyvitamin D₃ from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25-dihydroxyvitamin D₃, 7.25 nM and 72.5 nM, displaced 1α25-dihydroxyvitamin D₃ binding while 25-hydroxyvitamin D₃, 7.25 nM, did not. The apparent dissociation constant (K_D) for 1α25-dihydroxyvitamin D₃ was detrermined from displacement of ³H-1α25-dihydroxyvitamin D₃ yielding a value of 8.1 × 10^?7 M by Scatchard analysis. The K_D for the 1β epimer determine from displacement of ³H-1α25-dihydroxyvitamin D₃ was 4.8 × 10^?7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25-dihydroxyvitamin D₃ and its 1β epimer, but not 25-dihydroxyvitamin D₃. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.     

Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I,osteopontin, and osteocalcin gene expression in chicken osteoblasts

Seventeen day chicken embryonic osteoblasts treated over a 30-day period with 1,25(OH)₂ D₃ showed a 2–10-fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α₁(I) and α₂(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)₂ D₃ at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5-day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)₂ D₃ treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)₂ D₃ in primary avian osteoblast cultures derived from 17-day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.     

Characterization of HMBP-2, a DNA-binding protein that binds to HIV-1 LTR when only one of the three Sp1 sites is methylated

HIV-1 methylation binding protein-1 (HMBP-1) (formerly called HMBP) is a protein found in human cell nuclei that binds with enhanced affinity to a fragment of the HIV-1 long terminal repeat sequence (LTR) containing three Sp1 sites when all three sites are methylated. HMBP-2 is another protein present in the nuclei of human T helper lymphocytes and HeLa cells that binds to the HIV-1 LTR. HMBP-2 binds preferentially to the same region of the HIV-1 LTR as does HMBP-1, but HMBP-2 binds best when only one of the three Sp1 sites is methylated. HMBP-2 can be separated from HMBP-1 chromatographically, and dimethyl sulfate (DMS) methylation interference analysis indicates that their binding sites are not identical. HMBP-2 represents a novel protein factor capable of binding to a partially methylated region of the HIV-1 LTR.     

Effects of clinorotation on COL1A1-EGFP gene expression

Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of α 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.