Osteogenic Maturation and Bone Repair Require 1,25- and 24,25-dihydroxyvitamin D

成骨成熟和骨修复需要 1,25- 和 24,25-二羟基维生素 D

• 批准号: 8632190
• 负责人: Kevin Matthew Curtis
• 金额: $ 5.6万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-20 至 2016-05-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8632190
• 关键词: Osteogenic; Maturation; Bone; Repair; Require

DESCRIPTION (provided by applicant): The vitamin D endocrine system is fundamental to health maintenance and disease prevention. Bone homeostasis and repair is regulated by a network of vitamin D metabolites, of which 1¿,25-dihydroxyvitamin D (1,25OHD) is considered the most biologically active and relevant in bone health. Vitamin D is also metabolized into other forms, including 24,25-dihydroxyvitamin D (24,25OHD), which we believe is fundamental for human mesenchymal stem cell (hMSC) osteogenic maturation and bone formation. Our data establishes that 24,25OHD inhibits proliferation, increases alkaline phosphatase activity and mineralization, and decreases 1¿-hydroxylase expression, potentially decreasing the ability of hMSC to convert 25OHD to 1,25OHD, while promoting osteogenic maturation. hMSC also express a 24,25OHD receptor (24,25OHDR), suggesting direct actions of 24,25OHD, and further implicating 24,25OHD in osteogenic maturation. Currently, we do not understand how 24,25OHD and 1,25OHD differentially mediate this process. During murine development, the transcription factor p63 is required for skeletal formation, and our recent work demonstrated that p63 is integral for hepatocyte growth factor (HGF) and 1,25OHD induced VDR expression and osteogenic maturation. This demonstrates that p63 mediates 1,25OHD/VDR, and poses the question as to its regulation of 24,25OHD actions. p63 gene expression is complex, leading to the formation of TA- and ¿Np63 isoforms, and three splice variants (¿,¿,?). The TA/¿Np63 isoforms act in opposition to activate or repress growth and differentiation, however the biological significance of p63 splice variants (¿,¿,?) in osteogenic maturation and bone repair are not established. Our studies demonstrate up-regulation of Np63 isoforms by 1,25OHD and alteration of p63? by 24,25OHD, linking 1,25OHD up-regulation of Np63, required for terminal differentiation, and 24,25OHD up-regulation of p63¿, known to regulate VDR expression. Thus, p63 appears to be central in the modulation of bone homeostasis by 1,25OHD and 24,25OHD. We hypothesize that the actions of vitamin D on osteogenic maturation and bone repair are due to a regulatory relationship between p63 gene products and unique 24,25OHD and 1,25OHD effects. We will test this hypothesis by assessing the regulation of p63 gene products by 24,25OHD and 1,25OHD. We expect Np63 will increase with 1,25OHD, and 24,25OHD will increase the p63? variants. To confirm the requirement of p63 during this process, we will use stable shRNA lentiviral vectors to knock-down first the TA- and Np63 forms, followed by the splice variants (¿,¿,?) . We predict that ablation of ¿Np63? will block vitamin D induced effects. To test this hypothesis in vivo, we will use a rat xenograft model of bone repair. We predict that over-expression of ¿Np63? will increase the osteogenic maturation of hMSC, promoting bone repair. We expect to identify a variant of p63 as a major regulatory component of osteogenic maturation by 1,25OHD and 24,25OHD. Those results would support a generalized paradigm implicating p63 as a key player during hMSC differentiation and bone repair.

描述（由申请人提供）：维生素 D 内分泌系统是维持健康和预防疾病的基础，由维生素 D 代谢物网络调节，其中 1¿ ,25-二羟基维生素 D (1,25OHD) 被认为是最具生物活性且与骨骼健康相关的维生素 D，也可代谢为其他形式，包括 24,25-二羟基维生素 D (24,25OHD)，我们认为它对于骨骼健康至关重要。人类间充质干细胞 (hMSC) 成骨成熟和骨形成 我们的数据表明，24,25OHD 抑制增殖，增加碱性。磷酸酶活性和矿化，并降低 1¿ -羟化酶表达，可能降低 hMSC 将 25OHD 转化为 1,25OHD 的能力，同时促进成骨成熟。 hMSC 还表达 24,25OHD 受体 (24,25OHDR)，表明 24,25OHD 的直接作用，并进一步暗示 24, 25OHD 在成骨成熟中的作用 目前，我们尚不清楚 24,25OHD 和 1,25OHD 有何不同。在小鼠发育过程中，转录因子 p63 是骨骼形成所必需的，我们最近的工作表明，p63 是肝细胞生长因子 (HGF) 和 1,25OHD 诱导的 VDR 表达和成骨成熟的组成部分。 1,25OHD/VDR，并提出了其对 24,25OHD p63 基因表达的调节是复杂的问题，导致形成。 TA- 和 ¿ Np63 同工型和三个剪接变体 (¿、¿、?)。 Np63 亚型的作用与激活或抑制生长和分化相反，但是，p63 剪接变体 (¿,¿,?) 在成骨成熟和骨修复中的生物学意义尚未确定，我们的研究表明 Np63 亚型上调 1, 25OHD 和 p63 的 24,25OHD 改变，连接 1,25OHD 的 Np63 上调，这是终末分化所需的，以及p63 的 24,25OHD 上调¿ ，已知调节 VDR 表达，因此，p63 似乎是 1,25OHD 和 24,25OHD 调节骨稳态的核心，我们发现维生素 D 对成骨成熟和骨修复的作用是由于调节关系。 p63 基因产物与独特的 24,25OHD 和 1,25OHD 效应之间的关系 我们将通过评估 p63 基因产物的调节来检验这一假设。 24,25OHD 和 1,25OHD 会随着 1,25OHD 的增加而增加，并且 24,25OHD 会增加 p63? 变体在此过程中的需要，我们将使用稳定的 shRNA 慢病毒载体来敲除。首先是 TA- 和 Np63 形式，然后是剪接变体 (¿,¿,?) 我们预测消融。 ¿ Np63 会阻断维生素 D 诱导的作用 为了在体内验证这一假设，我们将使用大鼠异种移植模型来预测 ¿ Np63？将增加 hMSC 的成骨成熟，促进骨修复，我们期望通过 1,25OHD 和 24,25OHD 确定 p63 的变体作为成骨成熟的主要调节成分。 hMSC 分化和骨修复过程中的关键角色。