Ahr and Osteoporosis

描述(申请人提供)：吸烟者患有严重的骨质疏松症，因此有极高的骨折风险。然而，吸烟导致骨质流失的机制仍不清楚。我们发现，在香烟烟雾中发现的200多种已知化学物质中，BaP(苯并[a]芘)和TCDD(2，3，7，8-四氯二苯并-对二恶英)可以触发芳香烃受体(Ahr)和细胞色素P450(Cyp1)酶来刺激骨骼去除。尽管有这些观察，我们在理解上仍然存在一些差距。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作用。因此，在特定的目标1中，我们将研究AHR基因在每种细胞类型中选择性缺失的小鼠的骨表型，然后检测AHR激动剂BaP和TCDD对骨量和重塑的影响。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Cyp1的激活，以及由此产生的活性氧物种(ROS)是否介导了这一作用。因此，在特定的目标2中，我们将给予BaP和/或TCDD来敲入在线粒体或微粒体中表达Cyp1蛋白的小鼠。我们将对它们的骨骼进行表型鉴定，并研究在分离的骨髓细胞中产生ROS。我们以前的研究进一步表明，ROS激活线粒体到细胞核的信号，产生一个前破骨细胞足迹，包括CREB、C/EBPδ、NF-κB、NFAT2和hnRNPA2。在特定的目标3中，我们将确定这种转录足迹是否被AhR激动剂激活。如果是这样的话，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-报告分析和芯片来确认足迹反式激活破骨细胞基因的表达。这些研究应该建立AHR作为治疗骨质疏松症的靶点，并至少在一定程度上解开吸烟者骨质疏松的分子基础。

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.