Human Osteoprogenitor Control by Hepatocyte Growth Factor and Vitamin D

肝细胞生长因子和维生素 D 对人类骨祖细胞的控制

• 批准号: 8536083
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8536083
• 关键词: 1,25 (OH) vitamin D; Alternative Splicing; Binding; Biological; Biological Assay; Biology; Bone Development; Bone Marrow; Bone Regeneration; Cartilage; Cells; Chondrocytes; Chondrogenesis; Complement Factor D; Data; Defect; Degenerative polyarthritis; Diagnostic radiologic examination; Disease; Evaluation; Event; Family; Funding; Gene Activation; Gene Expression; Genes; Growth Factor; Hepatocyte Growth Factor; Human; Image; Introns; Knock-out; Lead; Length; Luciferases; Maintenance; Mediating; Mesenchymal Stem Cells; Messenger RNA; Methodology; Modality; Modeling; Modification; Nude Rats; Osteoblasts; Osteogenesis; Osteoporosis; Outcome Study; Pathway interactions; Play; Population; Process; Production; Protein Isoforms; RNA Splicing; Rattus; Receptor Gene; Regulation; Relative (related person); Response Elements; Role; Sampling; Skeleton; Small Interfering RNA; Stem cells; Tail; Techniques; Testing; Time; Tissue-Specific Gene Expression; Tissues; Transactivation; Up-Regulation; Validation; Variant; Veterans; Viral; Vitamin D; Vitamin D3 Receptor; Western Blotting; Work; Xenograft Model; base; bone; bone healing; bone metabolism; cartilage development; cartilage repair; cell type; digital; effective therapy; expectation; frailty; in vivo; lipid biosynthesis; member; novel therapeutics; osteoblast differentiation; osteogenic; osteoprogenitor cell; promoter; receptor; receptor expression; repaired; response; stem cell differentiation; stem cell therapy; transcription factor; vector; vertebra body

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.

描述（由申请人提供）： 本申请描述了由肝细胞生长因子 (HGF) 和 1,25-二羟基维生素 D (1,25OHD)（维生素 D 最活跃的代谢物）催化的人间充质干细胞 (MSC) 分化和成熟的拟议研究。数据显示，p63、转录因子 p53 家族的成员，在 HGF 和 1,25OHD 上调维生素 D 受体 (VDR) 的协同作用中发挥着重要作用初步数据表明，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改变。p63 亚型基因表达的调节涉及两个不同的启动子（上游启动子和替代启动子）。位于内含子 3) 和选择性剪接以生成 mRNA，具体取决于所选的启动子，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) deltaN-（缺少 NH2 末端的一部分）p63。这两种形式也有不同的 RNA 剪接变体，称为 TAp63- 或 deltaNp63a、B 和 y，主要取决于 C 末端的长度。 p63 可以相反地激活或抑制干细胞介导事件中 RNA 剪接变体的生物学意义尚不清楚。 (VDR) 是 MSC 分化的重要调节因子，1,25OHD（与 VDR 结合）激活 VDR 和 p63 基因表达，p63 与 VDR 启动子结合并上调 VDR 基因表达，HGF 刺激 VDR 表达和 HGF 调节。 MSC 成骨细胞分化的过程可以通过降低 p63 表达来阻断，因此可以重新认识到 MSC 分化的 1,25OHD + HGF 调节依赖于上游的转换。 p63 启动子（TA，阻遏蛋白）连接到内部 p63 启动子（deltaN，激活蛋白），介导骨和软骨发育。这种 1,25D/HGF 调节的 p63 开关导致 deltaN 形式与 TA 形式的增加，并且与 α 和 β 剪接变体相比，γ 剪接相对增加 为了检验这一假设，HGF、1,25OHD 和 HGF+1,25OHD 的影响变体。将使用荧光素酶测定来鉴定成骨细胞分化过程中 p63 启动子选择/激活（TA 与 deltaN）的变化，以确定启动子选择的变化。ChIP 测定将鉴定 1,25OHD 激活的 VDR 与 TA 与 deltaNp63 启动子上推定反应元件的特异性结合，以及鉴定与 VDR 启动子结合的 p63 亚型 鉴定响应 HGF 和 1,25OHD 的剪接变体的变化。将通过 RT-qPCR 和蛋白质印迹进行，然后进行 siRNA 敲低，然后通过特定变体的慢病毒稳定过表达来确认/验证特定 p63 亚型/变体在 MSC 介导的体内骨修复中的作用。使用已建立的无胸腺裸鼠骨修复“钻孔”异种移植模型，用于 TA- 和 TA- 和慢病毒过表达（OX）载体。 Np63 和特定目标 #1 中确定的特定变体将用于在 MSC 中产生 p63 变体的稳定过表达。该模型涉及在第三尾椎体中制作可重复的缺陷（钻孔），然后进行。在研究期（8-12 周）结束时，将 MSC（对 p63 进行各种修饰）放入孔中后，通过 X 射线图像的数字分析来量化骨愈合率随时间的变化。 ¿将对样本进行 CT 成像和免疫组织化学分析，以进一步明确观察到的变化，总体研究的第三部分将是评估/鉴定 HGF 诱导的 MSC 软骨形成和脂肪形成分化过程中的 p63 基因表达和激活效应，1。 ,25OHD 和 1,25OHD+HGF 使用上述技术进行成骨分化，预计这些研究将鉴定 p63 的特定形式/剪接变体作为 VDR 调节的主要成分。 1,25OHD 和 HGF，在 MSC 分化过程中，支持暗示 p63 在 MSC 分化过程中发挥关键作用的通用范式将进一步加强这一范式，但超出了本提案的范围。