Molecular and cellular determinants of enthesopathy in X-linked hypophosphatemia

 描述（由申请人提供）：项目摘要/摘要X连锁低磷酸盐血症（XLH）是遗传性佝偻病的最常见形式，其特征是FGF 23水平升高，导致血清磷酸盐降低和1，25二羟维生素D（1，25（OH）2D）产生受损。内肽酶PHEX的突变是XLH表型的原因。虽然已知FGF 23的增加是由PHEX突变引起的，但其分子基础仍然未知。附着点（肌腱-骨附着部位）的病理性矿化，称为附着点病变，是XLH的并发症。常见的受累部位包括跟腱和髌骨末端。虽然它会导致患者的显著发病率，但对引起末端病的分子机制以及目前可用的治疗方式对末端病发展的影响知之甚少。低磷酸盐血症的小鼠模型，包括维生素D受体敲除（VDR KO）和肾II型钠依赖性磷酸盐协同转运蛋白（Npt 2a）KO，不具有末端病。相比之下，低磷酸盐血症和血清FGF 23水平升高的小鼠，包括Hyp小鼠（XLH小鼠模型）和牙本质基质蛋白1（DMP 1）KO，确实发生了末端病，表明FGF 23而不是血清磷酸盐可能在末端病的发生中起致病作用。然而，VDR KO小鼠直到3周龄才发生佝偻病，而Npt 2aKO小鼠的佝偻病由于1，25（OH）2D水平增加而在4周内逆转。由于Hyp和DMP 1 KO小鼠从出生起就患有进行性佝偻病，骨骼矿化受损可能会改变成熟附着点的机械力，导致XLH和DMP 1失活中附着点病的发展。 提出的研究重点是阐明这种附着点病的小鼠模型XLH（Hyp小鼠）的分子和细胞基础。目的I中提出的研究将提供Hyp小鼠附着点异常细胞的分子特征，并确定与附着点病发展有关的信号通路。Aim II中的谱系追踪研究将确定Hyp附着点中这些细胞的起源及其异常分化的病理生理学基础。目的III中提出的研究将确定FGF 23、矿化受损和机械应变对肌腱端病发展和进展的相对贡献。这种异常矿化的分子病理生理学基础也将提供与其他肌肉骨骼疾病相关的附着点异常的见解。先前的研究主要集中在胚胎肌腱的发育，因此这些实验将有助于我们了解正常的出生后附着点成熟。 主要研究者刘博士是一名医生兼科学家，其长期目标是领导一个专注于肌肉骨骼生物学的独立基础科学实验室。她在研究降钙素对XLH患者血清FGF 23水平的影响时，对罕见骨病产生了浓厚的兴趣。她为这项研究设计、执行和进行了分析，并在《新英格兰医学杂志》上发表了第一作者论文。这项研究结合她在临床内分泌学方面的严格培训，使刘博士认识到临床经验如何为她的研究假设提供信息。她对研究XLH潜在的分子病理生理学的热情使她加入了骨骼生物学高级研究员玛丽德梅博士的实验室。刘博士获得了F32/NRSA资助，以研究不同的治疗方式，包括每日1，25（OH）2D和FGF 23阻断抗体，如何影响生长，矿物质离子稳态，软骨细胞分化和骨骼矿化。K 08奖将使刘博士能够利用哈佛骨骼生物学社区提供的丰富指导。她的共同导师Vicki罗森博士，咨询委员会和合作者，所有著名的研究人员在肌肉骨骼生物学，将为她提供宝贵的意见，实验设计和分析，以及职业指导。职业计划，包括与导师，课程工作，教学会议，研究研讨会和期刊俱乐部定期会议，旨在提高她的研究经验。刘博士将在设计、执行和解释旨在从分子和细胞水平解剖复杂骨骼表型的研究方面积累专业知识。她将完善她的赠款和手稿写作技巧，并沉浸在骨骼和肌腱生物学领域。精心设计的职业发展计划，加上严格的实验室经验，将为她提供必要的技能，她在学术机构建立她的独立研究计划。

 DESCRIPTION (provided by applicant): Project Summary/Abstract X-linked hypophosphatemia (XLH) is the most common form of inheritable rickets, characterized by elevated FGF23 levels leading to low serum phosphate and impaired production of 1,25 dihydroxyvitamin D (1,25(OH)2D). Mutations in the endopeptidase PHEX are responsible for the XLH phenotype. While it is known that increases in FGF23 result from PHEX mutations, the molecular basis for this remains unknown. Pathologic mineralization of the enthesis (tendon-bone attachment site), referred to as enthesopathy, is a complication of XLH. Common sites of involvement include the Achilles and patellar entheses. Although it causes significant morbidity in patients, the molecular mechanisms responsible for the enthesopathy and impact of currently available modalities of treatment on the development of enthesopathy are poorly understood. Murine models of hypophosphatemia, including the vitamin D receptor knockout (VDR KO) and the renal type II sodium dependent phosphate co-transporter (Npt2a) KO do not have enthesopathy. In contrast, mice with hypophosphatemia and elevated serum FGF23 levels, including the Hyp mouse (murine model of XLH) and the dentin matrix protein 1 (DMP1) KO, do develop enthesopathy, suggesting FGF23 and not serum phosphate could have a pathogenic role in enthesopathy development. However, VDR KO mice do not develop rickets until 3 weeks of age, while the rickets in Npt2aKO mice reverses by 4 weeks due to increased 1,25(OH)2D levels. Since both the Hyp and DMP1 KO mice have progressive rickets from birth, the impaired skeletal mineralization may alter mechanical forces in the maturing enthesis, contributing to the development of enthesopathy in XLH and DMP1 inactivation. The studies proposed are focused on elucidating the molecular and cellular basis for this enthesopathy in a murine model of XLH (Hyp mice). Investigations proposed in Aim I will provide a molecular characterization of the abnormal cells in the entheses of Hyp mice and identify signaling pathways implicated in the development of enthesopathy. The lineage tracing studies in Aim II will identify the origin of these cells in the Hyp enthesis and the pathophysiologic basis for their aberrant differentiation. The studies proposed in Aim III will identify the relative contributions of FGF23, impaired mineralization and mechanical strain on enthesopathy development and progression. The molecular pathophysiology underlying this aberrant mineralization will also provide insight into enthesis abnormalities associated with other musculoskeletal disorders. Prior studies have focused on embryonic tendon development, thus these experiments will contribute to our understanding of normal post-natal enthesis maturation. The PI, Dr. Liu, is a physician-scientist whose long-term goal is to lead an independent basic science laboratory with a focus on musculoskeletal biology. She developed a strong interest in rare bone diseases while examining the effect of calcitonin on serum FGF23 levels in patients with XLH. She designed, executed and performed analyses for this study, which led to a first author publication in the New England Journal of Medicine. This study combined with her rigorous training in clinical endocrinology allowed Dr. Liu to recognize how clinical experiences can inform her research hypotheses. Her passion for studying the molecular pathophysiology underlying XLH led her to join the laboratory of Dr. Marie Demay, a senior investigator in skeletal biology. Dr. Liu was awarded a F32/NRSA grant to investigate how different treatment modalities, including daily 1,25(OH)2D and FGF23 blocking antibody, affect growth, mineral ion homeostasis, chondrocyte differentiation, and skeletal mineralization. A K08 award will allow Dr. Liu to take advantage of the rich mentorship available in the Harvard skeletal biology community. Her co-mentor Dr. Vicki Rosen, advisory committee, and collaborators, all renowned investigators in musculoskeletal biology, will provide her with valuable advice on experimental design and analyses, as well as career guidance. The career plan, consisting of regular meetings with mentors, course work, didactic conferences, research seminars and journal clubs is designed to enhance her research experience. Dr. Liu will develop expertise in the design, execution and interpretation of studies aimed at dissecting complex skeletal phenotypes at the molecular and cellular levels. She will refine her grant and manuscript writing skills and immerse herself in the field of skeletal and tendon biology. The carefully designed career development plan, combined with a rigorous laboratory experience will provide her with the skills necessary for her to establish her independent research program in an academic institution.