p63 Regulation of Human Osteoprogenitor Maturation by Hepatocyte Growth Factor & Vitamin D Metabolites

p63 肝细胞生长因子对人类骨祖细胞成熟的调节

• 批准号: 9512550
• 负责人: Guy Howard
• 金额: --
• 依托单位: MIAMI VA HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9512550
• 关键词: Alternative Splicing; Animal Model; Binding; Biological; Biological Assay; Biology; Bone Development; Bone Diseases; Bone Marrow; Bone Regeneration; Cell Cycle Progression; Cell Maturation; Cell Proliferation; Cells; Chondrocytes; Clinical; Complement Factor D; Data; Defect; Degenerative polyarthritis; Dihydroxycholecalciferols; Disease; Event; Family; Fracture; Funding; Gene Expression; Genes; Growth; Growth Factor; HGF gene; Human; Image; Individual; Introns; LIF gene; Lead; Length; Liver; Luciferases; Maintenance; Mediating; Mediation; Mesenchymal Stem Cells; Messenger RNA; Metabolism; Methodology; Methods; Modality; Modeling; Modification; Molecular; Nude Rats; Osteoblasts; Osteoporosis; Peripheral Vascular Diseases; Phase II Clinical Trials; Plasmids; Play; Population; Process; Production; Protein Isoforms; Proteins; Publications; RNA Splicing; Rattus; Regulation; Reporting; Reproducibility; Response Elements; Role; Safety; Sampling; Signal Transduction; Skeleton; Small Interfering RNA; Stem cells; TP53 gene; Tail; Testing; Therapeutic; Tissue-Specific Gene Expression; Tissues; Transactivation; Treatment Factor; Up-Regulation; Validation; Variant; Veterans; Vitamin D; Vitamin D3 Receptor; Western Blotting; Work; Xenograft Model; base; bone; bone healing; cell type; chromatin immunoprecipitation; effective therapy; expression vector; frailty; in vivo; in vivo Model; inhibitor/antagonist; innovation; knock-down; member; novel therapeutics; osteoblast differentiation; osteogenic; osteoprogenitor cell; overexpression; promoter; receptor; receptor expression; response; stem cell differentiation; stem cell therapy; tissue repair; transcription factor; vertebra body

The proposed studies focus on human mesenchymal stem cell (MSC) differentiation and maturation regulated by hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD), the most active metabolite of vitamin D in facilitating bone repair. These studies will determine the involvement of endogenous HGF in regulating the 1,25OHD upregulation of its receptor VDR. Our publications and preliminary data show 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) ΔN-(lacks part of the NH2 terminus) p63. These forms also have RNA splice variants denoted as TAp63- or ΔNp63α, β, and γ, depending on the length of the C-terminus. The TA and ΔN forms of p63 can act in opposition to activate or repress specific activities. The biological significance of 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 hMSC differentiation is dependent upon a switch from the upstream p63 promoter (TA, repressor) to the internal p63 promoter (ΔN, activator) mediating bone development. This 1,25D/HGF regulated p63 switch results in increases in the ΔN 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 ΔN) during osteoblastic differentiation will be identified with luciferase assays to see changes in promoter selection. ChIP assays will identify specific binding of 1,25OHD-activated VDR to response elements on the TA vs ΔNp63 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 and specific inhibitor knockdown, and by lentiviral stable over-expression of specific variants. Confirmation/validation of the role of specific p63 isoforms/variants in MSC-mediated bone repair in vivo, will be done using an established “drill-hole” model of bone repair in athymic nude rats. We have developed lentiviral over- expression vectors for TA- and ∆Np63 and the specific variant(s) with which we have produced stable over- expression of p63 variants in MSC. The in vivo model involves a reproducible defect (drill hole) in the third tail vertebral body, and then quantifying bone healing by μCT imaging after MSCs (with various modifications to p63) are placed into the hole. Because HGF is relatively short-lived in vivo, recent studies confirm the safety and efficacy of using a naked-plasmid for HGF to provide effective concentrations HGF in vivo. This efficient transient delivery of exogenous HGF, leads to activation of growth-related signal transduction events and promotion of cell proliferation (but not cancer).This approach is now in phase II clinical trials for peripheral vascular disease. We will use this method of HGF delivery to facilitate increased bone repair together with hMSC as described. At the end of the study period (8-12 weeks) both µCT imaging and immunohistochemical analyses will be done on the samples to define the changes observed / bone formed in the drill hole.

提出的研究重点是人间充质干细胞（MSC）分化和成熟 受肝细胞生长因子（HGF）和1,25-二羟基乙他素D（1,25OHD）调节，最活跃 维生素D的代谢物在支撑骨修复中。这些研究将确定 内源性HGF在调节其接收器VDR的1,25OHD上调。我们的出版物和 初步数据表明，p53转录因子家族的成员p63在 HGF和1,25OHD的合作作用在上调维生素D受体（VDR）并促进MSC 分化。试验数据表明，合作效应是基于p63差异的改变 替代启动子选择和RNA剪接变化产生的基因表达产物。规定 p63同工型基因表达涉及两个不同的启动子（上游启动子和替代方 位于内含子3）中的启动子和替代剪接以产生mRNA。根据选定的发起人的不同， 产生2种不同的形式：1）TA-（含有NH2末端的反式激活结构域）p63和2）Δn-（缺乏 NH2末端的一部分）p63。这些形式的RNA剪接变体表示为TAP63-或ΔNP63α，β， 和γ，具体取决于C末端的长度。 p63的TA和ΔN形式可以与 激活或抑制特定的活动。在干细胞 - 中介事件尚不清楚。维生素D接收器（VDR）是MSC分化的重要调节剂。 1,25OHD（与VDR结合）激活VDR和p63基因表达，p63与VDR启动子结合，并且 上调VDR基因表达和HGF模拟VDR表达和MSC的HGF调节 通过降低p63表达，可以阻止成骨细胞分化。假设 HMSC分化的1,25OHD + HGF调节取决于从上游p63的转换 启动子（TA，副本）介导骨发育的内部p63启动子（ΔN，激活剂）。这 1,25d/hgf调控的p63开关导致Δn形式的增加（s）与ta形式的增加，并且相对增加 与alpha和beta剪接变体相比，在伽马剪接变体中。为了检验这一假设 p63启动子选择/激活（tavsΔn）在成骨细胞上的HGF，1,25OHD和HGF+1,25OHD 将通过荧光素酶测定法确定分化，以查看启动子选择的变化。 CHIP分析会 确定1,25OHD激活的VDR与TA与ΔNP63启动子上响应元件的特定结合，为 以及识别与VDR启动子结合的p63同工型。识别剪接变体变化中的变化 对HGF和1,25OHD的响应将由RT-QPCR和Western印迹进行，其次是siRNA和特定 抑制剂敲低，并通过慢病毒的稳定过表达特定变体的稳定过表达。确认/验证 特定p63同工型/变体在MSC介导的体内骨修复中的作用将使用 在裸露大鼠中建立了骨修复的“钻孔”模型。我们已经开发了慢病毒过度 TA-和∆NP63的表达向量以及我们产生稳定过度的特定变体 MSC中p63变体的表达。体内模型涉及第三尾的可重复缺陷（钻孔） 椎体，然后在MSC后通过μCT成像来量化骨骼愈合（对 p63）放入孔中。由于HGF在体内相对较短，因此最近的研究证实了安全性 以及使用裸质质剂使HGF在体内提供有效浓度HGF的有效性。这个高效 外源HGF的瞬时递送，导致与生长有关的信号转导事件的激活和 促进细胞增殖（但不是癌症）。这种方法现在正在II期临床试验中 血管疾病。我们将使用这种HGF递送方法来促进骨修复的增加 如所述HMSC。在研究期结束时（8-12周）µCT成像和免疫组织化学 将对样品进行分析，以定义钻孔中观察到的 /骨的变化。