Modeling Dentin by G Protein Coupled Receptor Signaling

描述（由申请人提供）：这是一份由加州大学旧金山分校牙医科学家Orapin Horst博士提交的K08指导临床科学家研究职业发展奖的拟议申请。该奖项将为Horst博士提供必要的支持和培训，使她能够接受指导，发展成为一名独立的研究者，从而实现她的长期目标，即创造基于细胞的疗法和组织工程方法，以再现正常发育的方式再生牙本质-牙髓复合体。她将有机会获得UCSF的许多机会来帮助她完成以下与她的职业发展相关的目标：1)学习发育生物学和G蛋白偶联受体（GPCR）信号传导学科；2)与具有基础到临床转化工作经验的导师一起体验跨学科研究；3)培养学术技能，成为优秀的研究导师和教师；4)培养资助和获得资金的技能；5)为未来的R01应用收集试点数据。为了继续实现这些目标，Horst博士提出了一项研究计划，将阐明牙上皮细胞和间充质祖细胞之间的信号事件，以诱导成牙细胞分化并控制牙本质-牙髓复合体的形成。她将通过gpcr的功能来研究这些信号事件，gpcr有可能引发或改变任何信号通路。在其他细胞和组织类型，特别是骨中也有相关的发现，但对GPCR在牙齿中的功能知之甚少。本K08将测试的中心假设是gpcr调节的信号分子调节成牙本质分化和牙本质基质形成。这一假设的具体目的如下：1)确定牙齿间充质细胞（包括成牙细胞和成牙细胞前）中gs偶联的GPCR信号如何控制牙本质的形成；2)确定牙齿上皮细胞中gs偶联的GPCR信号如何调节成牙细胞的分化。这些目标将通过新开发的矿化组织形成中g蛋白信号的转基因小鼠模型来实现。在I型胶原启动子驱动下，成牙细胞和成牙前细胞中gs偶联GPCR信号增加的转基因小鼠将用于特异性Aim 1，而在角蛋白5启动子驱动下，牙上皮细胞中gs偶联GPCR信号增加的转基因小鼠将用于特异性Aim 2。这些研究将确定GPCR信号通路与调节成牙本质发育和牙本质基质形成的其他机制之间的新相互作用。最终，这些数据将为一个跨学科研究项目和R01拨款申请奠定基础，以设计一种支持成牙细胞和牙髓的管状牙本质生理复合体来治疗蛀牙。从这项工作中获得的知识也将提高我们对遗传牙齿畸形和环境因素的概念性理解，这些因素需要适当的细胞-基质相互作用来支持牙本质和其他矿化组织的正常发育。拟议的职业发展和研究活动将在高度支持和研究密集的环境中进行。两位国际牙齿发育生物学专家Pamela DenBesten DDS， MS（主要导师）和Ophir Klein MD， PhD（共同导师）将指导Horst博士在上皮-间质相互作用和矿化组织形成方面的研究重点。GPCR信号传导的两位国际专家Bruce Conklin MD（联合导师，干细胞GPCR信号传导）和Robert Nissenson博士（联合导师，骨GPCR信号传导）将监督转基因小鼠模型的工作，并将他们与其他干细胞类型和矿化组织的丰富经验联系起来，以了解GPCR信号传导在牙齿干细胞和牙本质-牙髓复合物形成中的作用。导师都是加州大学旧金山分校新成立的Eli and Edythe Broad再生医学和干细胞研究中心的成员，该中心将作为培训机会（讲习班、研讨会、期刊俱乐部和静修）的中心资源，将发育、分子和细胞生物学的专业知识与本项目中提出的医学相关焦点结合起来。拟议的研究是协调的职业发展计划的一部分，通过研究、课程和辅导，使候选人成为一名杰出的、独立的牙医科学家。跨学科的指导团队将共同指导职业发展活动，并促进Horst博士从目前的研究经验到指导临床科学家发展阶段的自然发展，然后走向独立。

DESCRIPTION (provided by applicant): This proposed application for a K08 Mentored Clinical Scientist Research Career Development Award is being submitted by Dr. Orapin Horst, a dentist-scientist at the University of California, San Francisco. This award will provide the support and training necessary for Dr. Horst to receive the mentoring to develop into an independent investigator, whereupon she will pursue her long-term goal of creating cell-based therapies and tissue engineering approaches to regenerate the dentin-pulp complex in a manner recapitulating normal development. She will have access to the many opportunities of UCSF to help her accomplish the following goals related to her career development: 1) to learn the disciplines of developmental biology and G protein coupled receptor (GPCR) signaling; 2) to experience interdisciplinary research with mentors experienced in basic to clinical translational work; 3) to build academic skills to become an outstanding research mentor and faculty member; 4) to develop skills in grantsmanship and obtaining funding; and 5) to collect pilot data for a future R01 application. To continue her progress towards these goals, Dr. Horst proposes a research plan that will elucidate signaling events between dental epithelial and mesenchymal progenitor cells to induce odontoblast differentiation and control the formation of the dentin-pulp complex. She will study these signaling events through the function of GPCRs, which have the potential to elicit or modify essentially any signaling pathway. Related findings have been reported in other cell and tissue types, in particular bone, but very little is known about GPCR function in teeth. The central hypothesis that will be tested in this K08 is that GPCR-modulated signaling molecules regulate odontoblast differentiation and dentin matrix formation. This hypothesis is addressed by the following specific aims: 1) To determine how Gs-coupled GPCR signals in dental mesenchymal cells including odontoblasts and preodontoblasts control dentin formation, and 2) To determine how Gs-coupled GPCR signals from dental epithelial cells modulate odontoblast differentiation. These aims will be pursued using a newly developed transgenic mouse model of G-protein signaling in mineralized tissue formation. Transgenic mice with increased Gs-coupled GPCR signals in odontoblasts and preodontoblasts driven by the type I collagen promoter will be used in Specific Aim 1, while mice with increased Gs-coupled GPCR signals in dental epithelial cells driven by the keratin 5 promoter will be used in Specific Aim 2. These studies will identify new interactions between GPCR signaling pathways and other mechanisms regulating odontoblast development and dentin matrix formation. Ultimately, these data will lay a foundation for an interdisciplinary research program and a R01 grant application to engineer a physiologic complex of tubular dentin supporting odontoblasts and pulp to heal carious teeth. The knowledge from this work will also improve our conceptual understanding of genetic dental malformations and environmental factors required for proper cell-matrix interactions to support normal development of dentin and other mineralized tissues. The proposed career development and research activities will take place in highly supportive, research- intensive environments. Two international experts in the developmental biology of teeth, Pamela DenBesten DDS, MS (primary mentor) and Ophir Klein MD, PhD (co-mentor), will supervise Dr. Horst's research focus on epithelial-mesenchymal interactions and formation of mineralized tissues. Two international experts in GPCR signaling, Bruce Conklin MD (co-mentor, GPCR signaling in stem cells) and Robert Nissenson PhD (co- mentor, GPCR signaling in bone), will supervise work with the transgenic mouse models, and relate their vast experience with other stem cell types and mineralized tissues, to understand the role of GPCR signaling in dental stem cells and the formation of the dentin-pulp complex. The mentors are all members of the new Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research of UCSF, which will serve as a central resource for training opportunities (workshops, seminars, journal clubs, and retreats) in combining expertise in developmental, molecular, and cellular biology with medically relevant foci such as that proposed in this project. The proposed research is part of a coordinated career development plan to prepare the candidate to be an outstanding, independent dentist-scientist through research, coursework, and tutorials. The interdisciplinary mentorship team will mentor the career development activities together, and facilitate Dr. Horst's natural progression from current research experiences to the mentored clinical scientist development phase and then to independence.