NOVEL VITAMIN D TARGET GENES IN OSTEOBLASTS

说明(申请人提供)：1，25-二羟基维生素D3(1，25(OH)2D3)是维生素D内分泌系统的生物活性激素。它通过维生素D受体(VDR)通过增加肠道钙吸收和直接影响成骨细胞/破骨细胞活动来影响骨骼。这项研究计划的总体目标是检测1，25(OH)2D3和VDR在成骨细胞中的直接转录效应。为了实现这一目标，表达阵列发现了几个新的基因，它们是由1，25(OH)2D3在成骨细胞中诱导的。这项建议集中在两个1，25(OH)2D3诱导的影响成骨细胞生物学的基因，即信号素3B(SEMA3B)和CCAAT/增强子结合蛋白β(C/EBPB)。SEMA3B是一种分泌的黏附分子，可以抑制细胞的生长和迁移。初步研究表明，SEMA3B在体内成骨细胞中表达，在体外培养的原代成骨细胞中受1，25(OH)2D3的调节。影像研究显示，在成骨细胞中表达人SEMA3B的转基因小鼠(Ob-SEMA3B)获得的胫骨松质骨密度显著降低。这些研究是第一次解决SEMA3B在骨骼动态平衡中的功能。C/EBPB是成骨细胞中受1，25(OH)2D3显著上调的转录因子，与VDR协同作用激活成骨细胞基因表达。初步数据显示，C/EBPb可激活成骨细胞中SEMA3B启动子的活性。在这项建议中检验的一个主要假设是，VDR和C/EBPB是SEMA3B基因表达和维持正常骨骼内稳态所需的其他基因的有效调节因子。我们建议通过关注这些新的小鼠模型的持续特征来检验维生素D内分泌系统在骨骼动态平衡中的意义。具体地说，我们建议：1)确定Ob-SEMA3B转基因小鼠矿化缺陷的潜在基础；2)建立成骨细胞选择性基因敲除小鼠，以测试SEMA3B在体内的生理作用；3)在细胞和分子水平上检测VDR和C/EBPs对SEMA3B的调控。这些研究将为维生素D内分泌系统在骨骼动态平衡中的直接作用提供新的见解。

DESCRIPTION (provided by applicant): 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the bioactive hormone of the vitamin D endocrine system. It functions via the vitamin D receptor (VDR) to impact bone by increasing intestinal calcium absorption and by directly affecting osteoblast/osteoclast activity. The overall goal of this research proposal is to examine direct transcriptional effects of 1,25(OH)2D3 and VDR in osteoblasts. Toward this goal, expression arrays identified several novel genes that are induced by 1,25(OH)2D3 in osteoblastic cells. This proposal focuses on two 1,25(OH)2D3-induced genes that impact osteoblast biology, namely Semaphorin 3B (SEMA3B) and CCAAT/enhancer binding protein beta (C/EBPb). SEMA3B is a secreted adhesion molecule that inhibits cell growth and migration. Preliminary data show that SEMA3B is expressed in osteoblasts in vivo and is regulated by 1,25(OH)2D3 in primary osteoblasts in vitro. Imaging studies reveal a dramatic decrease in trabecular bone density of tibias obtained from a transgenic mouse expressing human SEMA3B in osteoblasts (Ob-SEMA3B). These studies are the first to address SEMA3B function in skeletal homeostasis. C/EBPb is transcription factor that is dramatically upregulated by 1,25(OH)2D3 in osteoblastic cells and it acts cooperatively with VDR to activate osteoblast gene expression. Preliminary data show that C/EBPb activates SEMA3B promoter activity in osteoblasts. A major hypothesis tested in this proposal is that VDR and C/EBPb are potent regulators of SEMA3B gene expression and of other genes that are required to maintain normal skeletal homeostasis. We propose to examine the significance of the vitamin D endocrine system in the skeletal homeostasis by focusing on the continued characterization of these new mouse models. Specifically, we propose to: 1) identify the underlying basis for the mineralization defect in the Ob- SEMA3B transgenic mouse; 2) develop an osteoblast selective knockout mouse to test the physiological role of SEMA3B in vivo; 3) examine the regulation of SEMA3B by VDR and C/EBPs at the cell and molecular levels. These studies will provide new insight into the direct actions of the vitamin D endocrine system in skeletal homeostasis.