Analysis of transcription enhancing factor for PTH receptor that controls bone metabolism

甲状旁腺激素（PTH）和PTH相关蛋白（PTHrP）通过与成骨细胞中表达的G蛋白偶联的PTH/PTHrP受体（PTH1R）结合而影响骨代谢。它们增加一种破骨细胞激活因子（RANKL）的表达，促进骨吸收。大鼠、小鼠和人类PTH1R基因包含4个5'非编码外显子（U1、U2、U3和U4）和3个启动子区域。外显子U1上游的类tata启动子称为P1启动子，外显子U3和U4上游的富含gc的启动子分别称为P2和P3启动子。在大鼠中，下游启动子（P2）在包括骨和肾在内的多种组织中都有活性，而上游启动子（P1）是组织特异性的，在肾脏和卵巢中显示活性。在本研究中，我们试图分析转录相关因子和转录增强因子对大鼠成骨细胞PTH1R启动子P2的作用机制。实验结果如下：位于大鼠PTH1R U3启动子(P2) -128/-2区域的GC…More box被用作增强子，可以被SM探针取代，即Sp1和MAZ结合序列。ST2细胞成骨分化12天后PTH1R mRNA的表达。另一方面，Sp1和MAZ mRNA在ST2细胞成骨分化的所有时期（0天后）均有表达。Sp1 mRNA在所有组织中表达。PTH1R mRNA的表达模式与MAZ mRNA的表达模式有很大不同。在UMR-106细胞中，PTH1R mRNA的表达可通过敲低Sp1或Maz来抑制。在UMR-106细胞中，PTH1R基因U3启动子活性通过敲低Sp1而被抑制。ST2细胞成骨分化12天后PTH1R mRNA的表达。另一方面，在ST2细胞成骨分化8天后，osterix mRNA表达。PTH1R mRNA的组织表达模式与osterix的表达模式非常相似。在UMR-106细胞中，PTH1R mRNA的表达通过敲低osterix而被抑制。UMR-106细胞中，PTH1R基因U3启动子活性不受骨甾体敲除的抑制。少

Parathyroid hormone (PTH) and PTH related protein (PTHrP) exert their effects on bone metabolism by binding to the G protein-coupled PTH/PTHrP receptor (PTH1R) expressed on osteoblasts. They increase expression one of the osteoclast activating factor (RANKL) and enhance bone resorption.The rat, mouse and human PTH1R genes contain four 5' noncoding exons (U1, U2, U3 and U4) and three promoter regions. A TATA-like promoter upstream of exon U1 is referred to as the P1 promoter and a GC-rich promoter upstream of exon U3 and U4 is called the P2 and P3 promoters respectively. In rats, the downstream promoter (P2) was active in a variety of tissues including bone and kidney, while the upstream promoter (P1) was tissue-specific, showing activity in the kidney and the ovary.In the present study, we attempted to analyze the mechanism of transcription related factor(s) and transcription enhancing factors on the promoter P2 of the PTH1R in the rat osteoblasts. The results were as follows.1. The GC … More box located in the -128/-2 region in the rat PTH1R U3 promoter (P2) was performed as an enhancer and could be replaced by the SM probe that was known as Sp1 and MAZ binding sequence.2. The PTH1R mRNA expressed after 12-day of osteoblastic differentiation in ST2 cells. On the other hands, both Sp1 and MAZ mRNA expressed in all period of osteoblastic differentiation (after 0-day) in ST2 cells.3. The Sp1 mRNA expressed in all tissues examined. A pattern of PTH1R mRNA expression was quite different from that of MAZ mRNA expression.4. The PTH1R mRNA expression was suppressed by a knockdown of Sp1 or Maz in UMR-106 cells.5. The PTH1R gene U3 promoter activities were suppressed by a knockdown of Sp1 in UMR-106 cells.6. The PTH1R mRNA expressed after 12-day of osteoblastic differentiation in ST2 cells. On the other hands, osterix mRNA expressed after 8-day of osteoblastic differentiation in ST2 cells.7. A pattern of tissue distribution of PTH1R mRNA expression was quite similar to that of osterix mRNA expression.8. The PTH1R mRNA expression was suppressed by a knockdown of osterix in UMR-106 cells.9. The PTH1R gene U3 promoter activities were not suppressed by a knockdown of osterix in UMR-106 cells. Less