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

说明（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。骨稳态和修复是由维生素D代谢物网络调节的，其中1,25 -二羟基维生素D （1,250 ohd）被认为是最具生物活性和与骨骼健康相关的。维生素D也被代谢成其他形式，包括24,25-二羟基维生素D (24,25ohd)，我们认为它是人类间充质干细胞（hMSC）成骨成熟和骨形成的基础。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，降低1¿-羟化酶表达，潜在地降低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），提示24,25ohd的直接作用，并进一步暗示24,25ohd参与成骨成熟。目前，我们还不清楚24,25ohd和1,250 ohd是如何区别调解这个过程的。在小鼠发育过程中，转录因子p63是骨骼形成所必需的，我们最近的研究表明，p63是肝细胞生长因子（HGF）和1,250 ohd诱导的VDR表达和成骨成熟的组成部分。这表明p63介导1,250 ohd /VDR，并提出了其调控24,250 ohd作用的问题。p63基因表达复杂，导致TA-和¿Np63异构体的形成，以及三个剪接变体（¿，¿，？）。TA/ Np63异构体的作用与激活或抑制生长和分化相反，然而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，1,250 ohd上调了Np63亚型，p63？24,25ohd，连接终端分化所需的Np63的1,25ohd上调和已知调节VDR表达的p63¿的24,25ohd上调。因此，p63似乎是1,250 ohd和24,25 ohd调节骨稳态的核心。我们假设维生素D对成骨成熟和骨修复的作用是由于p63基因产物与独特的24,25ohd和1,25ohd效应之间的调节关系。我们将通过评估24,25ohd和1,25ohd对p63基因产物的调控来验证这一假设。我们预计Np63会增加1,250 ohd，而24,250 ohd会增加p63？变体。为了确认p63在这一过程中的需求，我们将使用稳定的shRNA慢病毒载体首先敲除TA-和Np63形式，然后是剪接变体（¿，¿，？）． 我们预测¿Np63？会阻止维生素D引起的影响为了在体内验证这一假设，我们将使用大鼠异种骨移植修复模型。我们预测过度表达¿Np63？会促进hMSC成骨成熟，促进骨修复。我们希望鉴定出p63的一种变体，作为1,250 ohd和24,250 ohd的成骨成熟的主要调节成分。这些结果将支持p63在hMSC分化和骨修复过程中发挥关键作用的普遍范式。

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.