The Role of RECQL4 in Bone Development and Osteosarcoma

描述（申请人提供）：Rothmund-Thomson综合征（RTS）是一种由RECQL 4基因突变引起的多系统遗传性疾病。我们以前的研究表明，这种综合征的特点导致了一些观察这些患者的骨缺损。RTS患者有严重的骨骼异常，包括骨质减少、发育不良或融合骨和骨小梁缺损。他们也有一个显着增加的风险发展成骨肉瘤（OS），一个原发性恶性骨肿瘤，这是非常需要新的治疗方法。当RECQL 4突变时，这些骨状况增加，表明RECQL 4在正常骨发育和抑制OS中起重要作用。然而，RECQL 4 DNA解旋酶的确切功能以及它如何在正常骨细胞和骨肿瘤中发挥作用尚不清楚。我们已经通过酵母双杂交试验表明RECQL 4与PRICKLE 1蛋白相互作用，PRICKLE 1蛋白是已知在骨发育和癌症中重要的Wnt信号通路的成员。本提案的目的是在临床上以及分子和细胞水平上检查RECQL 4功能丧失的骨骼后果，以确定RECQL 4在骨骼中的作用机制。为了验证我们的假设，RECQL 4是一种骨稳态蛋白，通过Wnt信号通路发挥其发育和致瘤作用，我们提出了以下目标。在目标1中，我们将通过详细的临床评价（包括DXA扫描、生化骨转换标志物和IV钙标记研究，以评估骨形成和骨吸收）来表征RTS患者中RECQL 4功能丧失导致的人类骨骼表型。在目标2中，我们将产生成骨细胞特异性Recql 4条件性敲除（CKO）小鼠模型，以通过详细的表型和骨组织形态计量学分析来表征Recql 4缺陷小鼠中的骨骼缺陷和癌症表型。在目标3中，我们将探索RECQL 4功能丧失对Wnt信号传导的影响，使用体外β-连环蛋白活性测定以及Wnt和相关信号传导途径的基因和蛋白质表达分析。了解RTS患者的骨骼缺陷以及骨形成和再吸收方面的细胞事件将使我们能够确定RECQL 4参与的分子途径以及它如何与骨发育和肿瘤发生相关。这些研究的结果将指导我们治疗RTS患者，深入了解骨骼疾病的机制，并可能最终为治疗一般人群的OS和骨质疏松症提供更多靶向治疗的机会。 公共卫生相关性：我们项目的目标是研究RECQL 4基因通路在骨肉瘤和其他骨疾病如骨质疏松症的发展中的作用。我们将研究患有人类遗传性疾病（Rothmund-Thomson综合征）的患者沿着RECQL 4缺陷的小鼠模型，以了解骨骼发育不良的细胞和分子基础。这将使我们能够发现新的方法来改善普通人群中骨肉瘤和骨质疏松症的治疗。

DESCRIPTION (provided by applicant): Rothmund-Thomson syndrome (RTS) is a mutisystem genetic disorder caused by mutations in the RECQL4 gene. Our previous studies characterizing this syndrome have led to several observations regarding bone defects in these patients. RTS patients have severe skeletal abnormalities, including osteopenia, hypoplastic or fused bones, and trabecular defects. They also have a significantly increased risk for developing osteosarcoma (OS), a primary malignant bone tumor for which new therapies are greatly needed. These bone conditions are increased when RECQL4 is mutated, suggesting that RECQL4 plays an important function in normal bone development and in suppression of OS. However, the exact function the RECQL4 DNA helicase and how it exerts its effects in normal bone cells and in bone tumors are not known. We have shown through yeast two-hybrid assay that RECQL4 interacts with PRICKLE1 protein, a member of the Wnt signaling pathway known to be important in both bone development and cancer. The objective of this proposal is to examine the skeletal consequences of loss of RECQL4 function both clinically and at the molecular and cellular level in order to define the mechanism of RECQL4 action in bone. To test our hypothesis that RECQL4 is a bone homeostasis protein that exerts its developmental and tumorigenic effects through the Wnt signaling pathway, we propose the following aims. In Aim 1, we will characterize the human skeletal phenotype that results from loss of RECQL4 function in RTS patients through detailed clinical evaluation, including DXA scanning, biochemical bone turnover markers, and IV calcium labeling studies to assess bone formation and resorption. In Aim 2, we will generate an osteoblast-specific Recql4 conditional knockout (CKO) mouse model to characterize the skeletal defects and cancer phenotypes in Recql4 deficient mice through detailed phenotypic and bone histomorphometric analyses. In Aim 3, we will explore the effects of loss of RECQL4 function on Wnt signaling using in vitro assays of b-catenin activity and gene and protein expression analyses of the Wnt and related signaling pathways. Understanding the skeletal defects in RTS patients and the cellular events in terms of bone formation and resorption will allow us to define the molecular pathways in which RECQL4 participates and how it relates to bone development and tumorigenesis. Results of these studies will guide us in the treatment of RTS patients, provide insight into the mechanisms of bone disease, and may eventually provide opportunities for more targeted therapies for the treatment of OS and osteoporosis in the general population. PUBLIC HEALTH RELEVANCE: The goal of our project is to study the role of the RECQL4 gene pathway in the development of osteosarcoma and other bone diseases such as osteoporosis. We will study patients with a human genetic disorder (Rothmund-Thomson syndrome) along with mouse models of RECQL4 deficiency to understand the cellular and molecular basis of skeletal dysplasia. This will allow us to discover novel ways to improve the treatment of osteosarcoma and osteoporosis in the general population.