Vitamin D Receptor Coactivators 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1，25(OH)2D3诱导的影响成骨细胞生物学的基因，即脑膜瘤-1或MN1。MN1是一种转录因子，在核受体介导的转录过程中充当辅助激活因子。在小鼠中，MN1基因的缺失明显损害了膜内成骨，导致颅骨发育缺陷。这些数据显示了MN1在骨骼动态平衡中的重要生物学作用。我们的初步数据表明，MN1在许多成骨细胞系中都有表达，并且在体外和体内的成骨细胞中，MN1的表达受到1，25(OH)2D3的调节。MN1在成骨细胞中的异位表达抑制了成骨细胞的增殖，shRNA介导的MN1表达下调在体外减少了成骨细胞的分化标志物。这些研究首次解决了MN1在骨骼动态平衡中的作用。在这项建议中检验的一个主要假设是，VDR和1，25(OH)2D3是MN1基因表达和维持正常骨骼动态平衡所需的其他基因的有效调节因子。我们建议通过关注MN1在成骨细胞功能中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MN1在附件和中轴骨骼发育和维持中的意义，2)确定1，25(OH)2D3在成骨细胞中调控MN1基因转录的分子机制，3)确定MN1在1，25(OH)2D3激活的成骨细胞转录中的功能意义，以及4)确定MN1在控制成骨细胞增殖和分化中的作用。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，并改善对维持骨骼完整性所需的关键因素的理解。与公共健康相关：维生素D在骨骼和成骨细胞中的作用所涉及的分子细节在很大程度上是未知的。我们的工作假说表明，维生素D控制MN1共激活蛋白的表达，并影响成骨细胞的功能。这项建议的主要目的是为成骨细胞中的VDR-RXR异源二聚体定义新的靶基因。

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 one 1,25(OH)2D3-induced gene that impact osteoblast biology, namely Meningioma-1 or MN1. MN1 is a transcription factor that serves as a coactivator in nuclear receptor- mediated transcription. Deletion of the MN1 gene in mice markedly impairs intramembranous ossification leading to defects in cranial skeletal development. These data demonstrate an important biological role for MN1 in skeletal homeostasis. Our preliminary data show that MN1 is expressed in numerous osteoblastic cell lines and its expression is regulated by 1,25(OH)2D3 in osteoblastic cells in vitro and in vivo. Ectopic expression of MN1 in osteoblasts inhibits osteoblast proliferation and shRNA- mediated knock-down of MN1 expression reduces osteoblast differentiation markers in vitro. These studies are the first to address MN1 function in skeletal homeostasis. A major hypothesis tested in this proposal is that VDR and 1,25(OH)2D3 are potent regulators of MN1 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 skeletal homeostasis by focusing on the continued characterization of MN1 in osteoblast function. Specifically, we propose to: 1) determine the significance of MN1 in development and maintenance of the appendicular and axial skeletons, 2) determine the molecular mechanisms involved in the transcriptional regulation of the MN1 gene by 1,25(OH)2D3 in osteoblastic cells, 3) establish the functional significance of MN1 in 1,25(OH)2D3-activated transcription in osteoblastic cells, and 4) determine the role of MN1 in controlling osteoblast proliferation and differentiation. These studies will provide new insight into the direct actions of the vitamin D endocrine system in skeletal homeostasis and an improved understanding of key factors that are needed to maintain skeletal integrity. PUBLIC HEALTH RELEVANCE: The molecular details involved in vitamin D effects in bone and osteoblasts are largely unknown. Our working hypothesis states that vitamin D controls the expression of the MN1 coactivator protein and impacts osteoblast function. The main goal of this proposal is to define novel target genes for VDR-RXR heterodimers in osteoblasts.