NOVEL VITAMIN D TARGET GENES IN OSTEOBLASTS

描述（由申请人提供）：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/破骨细胞活性来影响骨骼。本研究计划的总体目标是研究1,25(OH)2D3和VDR在成骨细胞中的直接转录作用。为了实现这一目标，表达阵列鉴定了成骨细胞中由125 (OH)2D3诱导的几个新基因。本研究主要关注影响成骨细胞生物学的两个1,25(OH) 2d3诱导基因，即信号蛋白3B （SEMA3B）和CCAAT/增强子结合蛋白β (C/EBPb)。SEMA3B是一种分泌性粘附分子，可抑制细胞生长和迁移。初步数据显示，SEMA3B在体内成骨细胞中表达，在体外原代成骨细胞中受1,25(OH)2D3调控。影像学研究显示，从成骨细胞中表达人SEMA3B的转基因小鼠（Ob-SEMA3B）获得的胫骨小梁骨密度急剧下降。这些研究首次探讨了SEMA3B在骨骼稳态中的功能。C/EBPb是成骨细胞中被125 (OH)2D3显著上调的转录因子，与VDR协同激活成骨细胞基因表达。初步数据显示，C/EBPb在成骨细胞中激活SEMA3B启动子活性。本研究的一个主要假设是，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小鼠模型的持续表征来检查维生素D内分泌系统在骨骼稳态中的意义。具体而言，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敲除小鼠，在体内测试SEMA3B的生理作用；3)在细胞和分子水平上考察VDR和C/ ebp对SEMA3B的调控作用。这些研究将为维生素D内分泌系统在骨骼稳态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.