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连锁低磷血症(XLH)是最常见的遗传性软骨病，其特征是FGF23水平升高导致血清磷酸盐水平降低，1，25二羟基维生素D(1，25(OH)2D)的生成受阻。内肽酶PHEX的突变是XLH型的原因。虽然已知FGF23的增加是由PHEX突变引起的，但其分子基础仍不清楚。末端(腱-骨附着部位)的病理性矿化，称为末端病，是XLH的并发症。常见的受累部位包括跟腱和膝盖骨。虽然它在患者中引起了显著的发病率，但对导致终端病的分子机制以及目前可用的治疗方式对终端病发展的影响还知之甚少。低磷血症的小鼠模型，包括维生素D受体基因敲除(VDR KO)和肾脏II型钠依赖磷酸共转运体(Npt2a)KO没有终末病。相反，低磷血症和血清FGF23水平升高的小鼠，包括Hyp小鼠(XLH的小鼠模型)和牙本质基质蛋白1(DMP1)KO，确实会发生终末病，这表明FGF23而不是血清磷可能在终末病的发生中起致病作用。然而，VDR KO小鼠直到3周大时才会出现软骨病，而Npt2aKO小鼠的软骨病在4周后会逆转，这是因为1，25(OH)2D水平增加。由于Hyp和DMP1 KO小鼠从出生起就患有进行性软骨病，骨骼矿化受损可能会改变成熟成熟期的机械力，导致XLH和DMP1失活的终末病的发生。建议的研究集中在阐明这种终末病的分子和细胞基础在XLH(Hyp小鼠)的小鼠模型中。AIM I中提出的研究将提供Hyp小鼠末端异常细胞的分子特征，并确定与终末疾病发展有关的信号通路。AIM II中的谱系追踪研究将确定精子内这些细胞的来源以及它们异常分化的病理生理学基础。AIM III中建议的研究将确定FGF23、矿化受损和机械应变在终末病发生和进展中的相对贡献。这种异常矿化背后的分子病理生理学也将提供与其他肌肉骨骼疾病相关的末端异常的洞察。以前的研究主要集中在胚胎肌腱的发育上，因此这些实验将有助于我们理解出生后正常的末端成熟。刘博士是一名内科科学家，他的长期目标是领导一个独立的基础科学实验室，专注于肌肉骨骼生物学。在研究降钙素对XLH患者血清FGF23水平的影响时，她对罕见的骨骼疾病产生了浓厚的兴趣。她设计、执行和执行了这项研究的分析，这项研究的第一作者发表在《新英格兰医学杂志》上。这项研究再加上她在临床内分泌学方面的严格培训，让刘博士认识到临床经验是如何为她的研究假设提供依据的。她对研究XLH背后的分子病理生理学的热情促使她加入了玛丽·德迈博士的实验室，她是骨骼生物学的高级研究员。刘博士被授予F32/NRSA奖金，以研究不同的治疗方式，包括每日1，25(OH)2D和FGF23阻断抗体，如何影响生长、矿物质离子动态平衡、软骨细胞分化和骨骼矿化。K08奖项将允许刘博士利用哈佛骨骼生物学社区提供的丰富指导。她的合作导师Vicki Rosen博士、顾问委员会和合作者都是肌肉骨骼生物学领域的知名研究人员，他们将在实验设计和分析以及职业指导方面为她提供宝贵的建议。职业计划包括与导师的定期会议、课程作业、教学会议、研究研讨会和期刊俱乐部，旨在增强她的研究经验。刘博士将在设计、执行和解释旨在从分子和细胞层面剖析复杂骨骼表型的研究方面发展专业知识。她将完善她的拨款和手稿写作技能，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，将为她提供在学术机构建立独立研究项目所需的技能。

 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.