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: 8698316
• 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/8698316
• 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; 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; X-Ray Computed Tomography; 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）： 本申请描述了肝细胞生长因子（HGF）和维生素D最具活性的代谢物1，25-二羟基维生素D（1，25 OHD）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，p53家族的转录因子p63在HGF和1，25 OHD上调维生素D受体（VDR）和促进MSC分化的协同作用中起主要作用。试点数据表明，合作的影响是基于改变p63差异基因表达产物的选择性启动子和RNA剪接的变化。p63亚型基因表达的调控涉及两个不同的启动子（上游启动子和位于内含子3的替代启动子）和选择性剪接以产生mRNA。根据所选择的启动子，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）deltaN-（缺少部分NH 2末端）p63。这两种形式也具有不同的RNA剪接变体，表示为TAp 63-或deltaNp 63 a、B和y，主要取决于C-末端的长度。p63的TA和deltaN形式可以相反地起作用以激活或抑制特定活动。RNA剪接变异体在干细胞介导的事件中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。1，25 OHD（结合VDR）激活VDR和p63基因表达，p63结合VDR启动子并上调VDR基因表达，并且HGF刺激VDR表达和HGF调节MSC成骨细胞分化可通过降低p63表达来阻断。因此，假设1，25 OHD + HGF对MSC分化的调节依赖于从上游p63启动子（TA，阻遏物）到内部p63启动子（deltaN，激活物）的转换，从而介导骨和软骨发育。这种1，25 D/HGF调节的p63开关导致deltaN形式相对于TA形式的增加，以及与α和β剪接变体相比γ剪接变体的相对增加。为了检验这一假设，将使用荧光素酶测定法鉴定HGF，1，25 OHD和HGF+1，25 OHD对成骨细胞分化期间p63启动子选择/活化（TA vs deltaN）的影响，以确定启动子选择的变化。ChIP测定将鉴定1，25 OHD活化的VDR与TA相对于deltaNp 63启动子上的推定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。将通过RT-qPCR和蛋白质印迹，随后通过siRNA敲低，然后通过特异性变体的慢病毒稳定过表达来鉴定剪接变体响应HGF和1，25 OHD的变化。将使用无胸腺裸大鼠中已建立的骨修复“钻孔”异种移植模型来确认/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。用于TA-和Np 63的慢病毒过表达（OX）载体以及在特定目标#1中鉴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造可再现的缺损（钻孔），随后在将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。在研究期（8-12周）结束时，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。总体研究的第三部分将是使用上述成骨分化技术评价/鉴定HGF、1，25 OHD和1，25 OHD +HGF诱导的MSC软骨形成和脂肪形成分化过程中p63基因表达和活化效应。这些研究预期将鉴定p63的特定形式/剪接变体作为在MSC分化期间通过1，25 OHD和HGF调节VDR的主要组分，支持暗示p63作为MSC分化期间的关键参与者的一般化范例。分化成其他血统将进一步加强这一范式，但超出了本建议的范围。