Human Osteoprogenitor Control by Hepatocyte Growth Factor and Vitamin D

描述(由申请人提供)： 本申请描述了肝细胞生长因子(HGF)和维生素D最活跃的代谢物1，25-二羟基维生素D(1，25OHD)对人骨髓间充质干细胞(MSC)分化和成熟的催化研究。数据显示，P63是P53转录因子家族的成员，在HGF和1，25OHD上调维生素D受体(VDR)和促进MSC分化的协同作用中发挥重要作用。Pilot数据表明，协同效应是基于替代启动子选择和RNA剪接变化导致的p63差异基因表达产物的变化。P63异构体基因表达的调控涉及两个不同的启动子(一个上游启动子和一个位于内含子3的交替启动子)和选择性剪接以产生mRNA。根据所选启动子的不同，会产生两种不同的形式：1)TA-(包含NH2末端的反式激活结构域)P63和2)Deltan-(缺少部分NH2末端)P63。这两种形式也有不同的RNA剪接变体，表示为TAp63-或deltaNp63a、B和y，主要取决于C-末端的长度。P63的TA和Deltan形式可以相反地作用于激活或抑制特定的活动。在干细胞介导的事件中，RNA剪接变体的生物学意义尚不清楚。维生素D受体(VDR)是MSC分化的重要调节因子。1，25OHD(与VDR结合)可同时激活VDR和P63基因的表达，P63结合VDR启动子并上调VDR基因的表达，而HGF对VDR表达的刺激和HGF对MSC成骨细胞分化的调控可以通过降低p63的表达来阻断。因此，1，25OHD HGF对MSC分化的调控依赖于从上游p63启动子(TA，抑制子)到内部p63启动子(Deltan，激活子)的转换，以介导骨和软骨的发育。这种受1，25D/HGF调控的P63开关导致Deltan形式(S)比TA形式(S)增加，并且与α和β剪接变异体相比，伽马剪接变异体相对增加。为了验证这一假说，我们将通过荧光素酶分析来确定HGF、1，25OHD和HGF 1，25OHD在成骨细胞分化过程中对p63启动子选择/激活(TA和Deltan)的影响，以确定启动子选择的变化。芯片分析将确定1，25OHD激活的VDR与TA和DeltaNp63启动子上可能的反应元件的特异性结合，以及p63亚型(S)与VDR启动子的结合。对HGF和1，25OHD的剪接变体的变化将通过RT-qPCR和Western blots进行鉴定，然后是siRNA敲除，然后是特定变体的慢病毒稳定过表达。确认/验证特异性p63异构体/变异体在体内MSC介导的骨修复中的作用，将使用已建立的裸鼠骨修复的“钻孔”异种移植模型来完成。针对TA-和Np63的Lenti病毒过表达(OX)载体和在特定目的#1中确定的特定变异体(S)将被用于在MSC中产生稳定的过表达P63变异体。该模型包括在第三个尾椎体制造一个可复制的缺陷(钻孔)，然后在MSCs(对P63进行各种修改)被植入洞中后，通过对X射线图像的数字分析来量化随着时间的推移骨愈合率。在研究结束时(8-12周)，将对样本进行CT成像和免疫组织化学分析，以进一步确定观察到的变化。整个研究的第三部分将利用上述成骨分化技术评估/鉴定HGF、1，25OHD和1，25OHD HGF诱导MSC向软骨和成脂分化过程中p63基因的表达和激活效应。这些研究有望确定p63的一个特定形式/剪接变体是1，25OHD和HGF在MSC分化过程中调节VDR的主要成分，支持一个普遍的范式，暗示P63在MSC分化过程中发挥关键作用。分化成其他血统将进一步加强这一范式，但超出了这一提议的范围。

DESCRIPTION (provided by applicant): This application describes proposed studies on human mesenchymal stem cell (MSC) differentiation and maturation catalyzed by hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD), the most active metabolite of vitamin D. Data shows that p63, a member of the p53 family of transcription factors, plays a major role in the cooperative actions of HGF and 1,25OHD to up-regulate the vitamin D receptor (VDR) and promote MSC differentiation. Pilot data suggests that the cooperative effects are based on alterations of p63 differential gene expression products resulting from alternative promoter selection and RNA splicing changes. Regulation of p63 isoform gene expression involves two distinct promoters (an upstream promoter and an alternate promoter located in intron 3) and alternative splicing to generate mRNA. Depending on the promoter selected, 2 distinct forms are produced: 1) TA-(transactivation domain containing NH2 terminus) p63 and 2) deltaN-(lacks part of the NH2 terminus) p63. These two forms also have distinct RNA splice variants denoted as TAp63- or deltaNp63a, B, and y, depending mainly on the length of the C-terminus. The TA and deltaN forms of p63 can act in opposition to activate or repress specific activities. The biological significance o the RNA splice variants during stem-cell-mediated events is not clear. The vitamin D receptor (VDR) is an important regulator of MSC differentiation. 1,25OHD (bound to VDR) activates both VDR and p63 gene expression, p63 binds to the VDR promoter and up-regulates VDR gene expression, and HGF stimulation of VDR expression and HGF regulation of MSC osteoblastic differentiation can be blocked by decreasing p63 expression. Thus it is hypothesized that 1,25OHD + HGF regulation of MSC differentiation is dependent upon a switch from the upstream p63 promoter (TA, repressor) to the internal p63 promoter (deltaN, activator) mediating bone and cartilage development. This 1,25D/HGF regulated p63 switch results in increases in the deltaN form(s) vs TA form(s), and a relative increase in gamma splice variants compared to alpha and beta splice variants. To test this hypothesis the effects of HGF, 1,25OHD and HGF+1,25OHD on p63 promoter selection/activation (TA vs deltaN) during osteoblastic differentiation will be identified using luciferase assays to determine changes in promoter selection. ChIP assays will identify specific binding of 1,25OHD activated VDR to putative response elements on the TA vs deltaNp63 promoters, as well as identify p63 isoform(s) binding to the VDR promoter. Identification of changes in splice variants in response to HGF and 1,25OHD will be done by RT-qPCR and western blots, followed by siRNA knockdown and then by lentiviral stable over-expression of specific variants. Confirmation/validation the role of specifi p63 isoforms/variants in MSC-mediated bone repair in vivo, will be done using an established "drill-hole" xenograft model of bone repair in athymic nude rats. Lenti-viral over- expression (OX) vectors for TA- and Np63, and the specific variant(s) identified in Specific Aim #1 will be used to produce stable over-expression of p63 variants in MSC. The model involves making a reproducible defect (drill hole) in the third tail vertebral body, and subsequently quantifying the rate of bone healing over time by digital analysis of X-ray images after MSCs (with various modifications to p63) have been placed into the hole. At the end of the study period (8-12 weeks) both ¿CT imaging and immunohistochemical analyses will be done on the samples to further define the changes observed. The third part of the overall studies will be an evaluation / identification of the p63 gene expression and activation effects during MSC chondrogenic and adipogenic differentiation induced by HGF,1,25OHD, and 1,25OHD+HGF using the techniques described above for osteogenic differentiation. These studies are expected to identify a specific form / splice variant of p63 as a major component of the regulation of VDR by 1,25OHD and HGF, during MSC differentiation, supporting a generalized paradigm implicating p63 as a key player during MSC differentiation. Differentiation into other lineages would further strengthen this paradigm, but are beyond the scope of this proposal.