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

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。骨稳态和修复由维生素D代谢物网络调节，其中1，25-二羟基维生素D（1，25 OHD）被认为是最具生物活性和与骨健康相关的。维生素D也代谢成其他形式，包括24，25-二羟基维生素D（24，25 OHD），我们认为这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，24，25 OHD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25 OHD转化为1，25 OHD的能力，同时促进成骨成熟。hMSC还表达24，25 OHD受体（24，25 OHDR），表明24，25 OHD的直接作用，并进一步暗示24，25 OHD在成骨成熟中。目前，我们尚不清楚24，25 OHD和1，25 OHD如何差异介导这一过程。在小鼠发育过程中，转录因子p63是骨骼形成所需的，我们最近的工作表明，p63是肝细胞生长因子（HGF）和1，25 OHD诱导的VDR表达和成骨成熟所必需的。这表明p63介导1，25 OHD/VDR，并提出了关于其调节24，25 OHD作用的问题。p63基因的表达是复杂的，导致TA-和<$Np63亚型的形成，以及三种剪接变体（<$，<$，？）。TA/？Np 63异构体的作用与激活或抑制生长和分化相反，然而，p63剪接变体的生物学意义（？，？，？）在成骨成熟和骨修复中没有建立。我们的研究表明上调Np 63亚型的1，25 OHD和改变p63？通过24，25 OHD，连接Np 63的1，25 OHD上调，这是终末分化所需的，以及已知调节VDR表达的p63的24，25 OHD上调。因此，p63似乎是1，25 OHD和24，25 OHD调节骨稳态的中心。我们假设维生素D对成骨成熟和骨修复的作用是由于p63基因产物与独特的24，25 OHD和1，25 OHD效应之间的调节关系。我们将通过评估24，25 OHD和1，25 OHD对p63基因产物的调节来验证这一假设。我们预计Np 63将增加与1，25 OHD，和24，25 OHD将增加p63？变体。为了证实在这个过程中p63的需要，我们将使用稳定的shRNA慢病毒载体首先敲低TA-和Np 63形式，然后敲低剪接变体（？，？，？）.我们预测，消融的<$Np63？会阻止维生素D的诱导作用为了在体内验证这一假设，我们将使用大鼠异种骨修复模型。我们预测，过度表达的<$Np63？将增加hMSC的成骨成熟，促进骨修复。我们希望通过1，25 OHD和24，25 OHD鉴定p63的变体作为成骨成熟的主要调节组分。这些结果将支持一个广义的范式，暗示p63作为一个关键的球员在hMSC分化和骨修复。

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.