Ahr and Osteoporosis

描述（由申请人提供）：吸烟者患有严重的骨质疏松症，因此骨骼骨折的风险极高。然而，吸烟导致骨质流失的机制仍不清楚。我们发现BaP（苯并[a]芘）和TCDD（2，3，7，8-四氯二苯并-p-二恶英），香烟烟雾中发现的200多种已知化学物质中的两种，触发芳烃受体（Ahr）和细胞色素P450（Cyp 1）酶，刺激骨去除。尽管有这些意见，我们的理解仍然存在一些差距。首先，我们不确定哪种骨细胞，破骨细胞，成骨细胞或骨细胞，主要介导这一行动。因此，在特定目标1中，我们将研究在每种细胞类型中选择性删除Ahr基因的小鼠的骨表型，然后检查Ahr激动剂BaP和TCDD对骨骼质量和重塑的影响。其次，我们不清楚Ahr激动剂的破骨细胞刺激作用是否涉及线粒体或微粒体Cyp 1的激活，以及如此产生的活性氧（ROS）是否介导了这一作用。因此，在具体目标2中，我们将给予BaP和/或TCDD基因敲入小鼠，在线粒体或微粒体中表达Cyp 1蛋白。我们将对它们的骨骼进行表型分析，并研究离体骨髓细胞中ROS的产生。我们以前的研究进一步表明，ROS激活骨细胞到细胞核的信号，产生包括CREB、C/EBPδ、NF-κB、NFAT 2和hnRNPA 2的促破骨细胞生成足迹。在特定目标3中，我们将确定这种转录足迹是否被Ahr激动剂激活。如果是这样，我们将利用siRNA和/或化学抑制剂来验证每个分子的作用，以及启动子-报告基因测定和ChIP来确认足迹反式激活破骨细胞基因表达。这些研究应确立Ahr作为骨质疏松症的治疗靶点，并至少在一定程度上揭示吸烟者骨质疏松症的分子基础。

DESCRIPTION (provided by applicant): Cigarette smokers suffer from severe osteoporosis, and are therefore at an exceptionally high risk of skeletal fracture. However, the mechanism through which smoke causes bone loss remains unclear. We find that BaP (benzo[a]pyrene) and TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), two of over 200 known chemicals found in cigarette smoke, trigger the aryl hydrocarbon receptor (Ahr) and the cytochrome P450 (Cyp1) enzymes to stimulate bone removal. Despite these observations, several gaps in our understanding remain. First, we are uncertain which bone cell, osteoclast, osteoblast or osteocyte, primarily mediates this action. Therefore, in Specific Aim 1, we will study the bone phenotype of mice in which the Ahr gene is deleted selectively in each cell type, and then examine the effect of the Ahr agonists BaP and TCDD on skeletal mass and remodeling. Second, we are unclear whether the osteoclast-stimulatory action of Ahr agonists involves the activation of mitochondrial or microsomal Cyp1s, and whether the reactive oxygen species (ROS) so produced mediate this action. Therefore, in Specific Aim 2, we will administer BaP and/or TCDD to knock-in mice expressing Cyp1 proteins either in mitochondria or in microsomes. We will phenotype their skeletons and study ROS production in isolated bone marrow cells. Our previous studies have further shown that ROS activate mitochondria-to-nucleus signals to generate a pro-osteoclastogenic footprint comprising CREB, C/EBPδ, NF-κB, NFAT2, and hnRNPA2. In Specific Aim 3, we will determine whether this transcriptional footprint is activated by Ahr agonists. If so, we will utilize siRNA and/or chemical inhibitors to validate the role of each molecule, as well as promoter-reporter assays and ChIP to confirm that the footprint transactivates osteoclast gene expression. These studies should establish the Ahr as a therapeutic target for osteoporosis and unravel, at least to an extent, the molecular basis underlying the osteoporosis noted in smokers.