The Role of RECQL4 in Bone Development and Osteosarcoma

描述（由申请人提供）：罗斯蒙-汤姆森综合征（RTS）是一种由RECQL4基因突变引起的多系统遗传疾病。我们之前的研究表征了这种综合征，导致了对这些患者的骨缺陷的一些观察。RTS患者有严重的骨骼异常，包括骨质减少、骨发育不良或融合，以及骨小梁缺陷。他们发生骨肉瘤（OS）的风险也显著增加，骨肉瘤是一种急需新疗法的原发性恶性骨肿瘤。当RECQL4突变时，这些骨骼状况增加，表明RECQL4在正常骨骼发育和抑制OS中起重要作用。然而，RECQL4 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。我们通过酵母双杂交实验表明，RECQL4与PRICKLE1蛋白相互作用，而PRICKLE1蛋白是Wnt信号通路的一个成员，已知在骨骼发育和癌症中都很重要。本提案的目的是在临床和分子和细胞水平上检查RECQL4功能丧失对骨骼的影响，以确定RECQL4在骨骼中的作用机制。为了验证我们的假设，即RECQL4是一种通过Wnt信号通路发挥其发育和致瘤作用的骨稳态蛋白，我们提出以下目标。在Aim 1中，我们将通过详细的临床评估，包括DXA扫描、生化骨转换标志物和静脉钙标记研究来评估骨形成和吸收，来描述RTS患者RECQL4功能丧失所导致的人类骨骼表型。在Aim 2中，我们将建立一个成骨细胞特异性Recql4条件敲除（CKO）小鼠模型，通过详细的表型和骨组织形态学分析来表征Recql4缺陷小鼠的骨骼缺陷和癌症表型。在Aim 3中，我们将通过体外b-连环蛋白活性分析和Wnt及相关信号通路的基因和蛋白表达分析，探讨RECQL4功能丧失对Wnt信号传导的影响。了解RTS患者的骨骼缺陷以及骨形成和骨吸收方面的细胞事件将使我们能够确定RECQL4参与的分子途径以及它与骨发育和肿瘤发生的关系。这些研究结果将指导我们对RTS患者的治疗，提供对骨病机制的深入了解，并可能最终为治疗普通人群的OS和骨质疏松症提供更有针对性的治疗方法。

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.