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.

本研究主要集中在人骨髓间充质干细胞（MSC）的分化和成熟 由肝细胞生长因子（HGF）和1，25-二羟维生素D（1，25 OHD）调节， 维生素D的代谢产物促进骨修复。这些研究将确定参与 内源性HGF在调节1，25 OHD上调其受体VDR中的作用。我们的出版物和 初步数据显示，p63，转录因子p53家族的成员，在细胞凋亡中起主要作用。 HGF和1，25 OHD协同作用上调维生素D受体（VDR），促进MSC 分化初步数据表明，协同效应是基于p63差异的改变， 基因表达产物由选择性启动子选择和RNA剪接改变产生。调控 p63亚型基因表达的调控涉及两个不同的启动子（上游启动子和替代启动子）。 启动子位于内含子3）和可变剪接以产生mRNA。取决于所选择的启动子， 产生2种不同的形式：1）TA-（含有NH 2末端的反式激活结构域）p63和2）Δ N-（缺乏 NH2末端的一部分）p63。这些形式也具有RNA剪接变体，表示为TAp63-或Δ Np63 α，β， 和γ，取决于C-末端的长度。p63的TA和Δ N形式可以与 激活或抑制特定活动。RNA剪接变异体在干细胞分化中的生物学意义 中介事件不明确。维生素D受体（VDR）是MSC分化的重要调节因子。 1，25 OHD（与VDR结合）激活VDR和p63基因表达，p63与VDR启动子结合， 上调VDR基因表达，HGF刺激VDR表达和HGF调节MSC 成骨细胞分化可通过降低p63表达而被阻断。因此，假设， 1，25OHD + HGF对hMSC分化的调控依赖于上游p63的转换 启动子（TA，阻遏物）与内部p63启动子（Δ N，激活物）之间的相互作用，介导骨发育。这 1，25D/HGF调节的p63开关导致Δ N形式相对于TA形式的增加，以及相对增加的Δ N形式。 与α和β剪接变异体相比，γ剪接变异体中的突变率更高。为了验证这一假设， HGF、1，25OHD和HGF +1，25OHD对成骨细胞中p63启动子选择/激活（TA vs Δ N）的影响 用荧光素酶测定鉴定分化以观察启动子选择的变化。ChIP检测将 鉴定1，25 OHD激活的VDR与TA vs Δ Np63启动子上的应答元件的特异性结合， 以及鉴定与VDR启动子结合的p63同种型。剪接变异体的鉴定 将通过RT-qPCR和蛋白质印迹，随后通过siRNA和特异性PCR， 抑制剂敲低，以及通过特异性变体的慢病毒稳定过表达。确认/验证 特异性p63同种型/变体在体内MSC介导的骨修复中的作用将使用 建立无胸腺裸大鼠"钻孔"骨修复模型。我们已经研制出了慢病毒- TA-和p53 Np63的表达载体以及我们用其产生稳定的过表达的特异性变体。 MSC中p63变体的表达。体内模型涉及第三条尾部的可再现缺陷（钻孔） 椎体，然后在MSC后通过μ CT成像定量骨愈合（对MSC进行各种修改以 p63）被放入孔中。由于HGF在体内的寿命相对较短，最近的研究证实了其安全性。 以及使用HGF裸质粒在体内提供有效浓度HGF的功效。这种高效 外源性HGF的瞬时递送导致生长相关信号转导事件的激活， 促进细胞增殖（但不是癌症）。这种方法现在正在进行外周血淋巴细胞的II期临床试验。 血管疾病我们将使用这种方法的肝细胞生长因子的交付，以促进增加骨修复连同 如所述的hMSC。在研究期结束时（8 - 12周）， 将对样本进行分析，以确定观察到的变化/钻孔中形成的骨。