Positive and negative control of the expression of parathyroid hormone (PTH)/PTH-related protein receptor via proximal promoter P3 in human osteoblast-like cells

Three promoters, P1, P2, and P3, regulate the expression of the receptor for the human PTH/PTH -related protein. The P3 promoter, proximal to the gene, seems to be turned on in many tissues and to be the most active of the three in the human adult kidney. P3 is also active in human osteoblastic SaOS-2 cells. Its structure to function relationship is, however, still poorly understood. To address this issue we assayed, in transiently transfected SaOS-2 cells, the expression of reporter gene constructs containing truncated P3 promoter fragments and substitution mutants. We thus localized cis-acting elements essential for P3 promoter activity and identified two key Sp1 binding sites. We also found in the 5'-untranslated exon U4, transcribed from promoter P3, an element that inhibits the expression of the receptor and is not promoter specific. This study provides new insights into PTH receptor expression in human osteoblast-like cells.     

Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.

Based on our previous transgenic mice results, which strongly suggested that separate cell-specific cis-acting elements of the mouse pro-alpha 1(I) collagen promoter control the activity of the gene in different type I collagen-producing cells, we attempted to delineate a short segment in this promoter that could direct high-level expression selectively in osteoblasts. By generating transgenic mice harboring various fragments of the promoter, we identified a 117-bp segment (-1656 to -1540) that is a minimal sequence able to confer high-level expression of a lacZ reporter gene selectively in osteoblasts when cloned upstream of the proximal 220-bp pro-alpha 1(I) promoter. This 220-bp promoter by itself was inactive in transgenic mice and unable to direct osteoblast-specific expression. The 117-bp enhancer segment contained two sequences that appeared to have different functions. The A sequence (-1656 to -1628) was required to obtain expression of the lacZ gene in osteoblasts, whereas the C sequence (-1575 to -1540) was essential to obtain consistent and high-level expression of the lacZ gene in osteoblasts. Gel shift assays showed that the A sequence bound a nuclear protein present only in osteoblastic cells. A mutation in the A segment that abolished the binding of this osteoblast-specific protein also abolished lacZ expression in osteoblasts of transgenic mice.     

非诺贝特与过氧化体增殖物激活型受体α对人肝瘤细胞1型纤溶酶原激活物抑制剂表达的影响

目的探讨非诺贝特对人肝瘤细胞株(HepG2)细胞1型纤维酶原激活物抑制剂(PAI-1)表达的影响及机制。方法用不同浓度非诺贝特刺激HepG2细胞,采用半定量逆转录聚合酶链反应(RT-PCR)法检测PAI-1mRNA水平,发色底物法检测PAI-1的活性变化。构建4个荧光素酶报告基因质粒,分别由PAI-1启动子序列从-804至+17间不同长度片段驱动,体外转染HepG2细胞,检测荧光素酶的活性。结果非诺贝特能使HepG2细胞PAI-1mRNA表达及蛋白活性显著降低,且呈一定剂量依赖性;还可使PAI-1转录活性显著降低;当转染质粒含有PAI-1启动子序列-636~+17、-449~+17-、276~+17 bp 3个片段时,荧光素酶活性显著增高;共转染过氧化体增殖物激活型受体α(PPARα)表达质粒(PPAR-αpSG5)的细胞在非诺贝特诱导下PAI-1转录活性显著降低。结论非诺贝特可以抑制HepG2细胞PAI-1mRNA表达及其活性,调节PAI-1的基因转录,PPARα参与非诺贝特对PAI-1基因的表达调控。