Role of TRPM4 in dental follicle stem cell differentiation

描述（申请人提供）：颅面组织功能障碍或缺陷对患者的生活有重大影响。因此，高度期望受损组织的重建。牙囊干细胞是组织替代的潜在来源，因为它们能够分化成专门的细胞，包括骨骼和脂肪（1）。然而，决定分化途径的基本过程仍然没有确定。因此，我们的长期目标是表征控制干细胞分化的分子机制，以便可以建立更严格和可靠的方案来重建颅面组件。钙（Ca 2+）振荡是干细胞分化过程中经常观察到的现象，有证据表明它可能对指导和终止该过程很重要（2-4）。瞬时受体电位Melastatin 4（TRPM 4）通道对Ca 2+振荡至关重要，因为它控制进入细胞的Ca 2+量（5）。TRPM 4的分子抑制增加免疫细胞中的Ca 2+进入和细胞因子产生（5）。事实上，增强的Ca 2+进入也有利于干细胞分化为骨和脂肪（2，3）。我们已经使用分子方法鉴定了干细胞中的TRPM 4，并使用膜片钳技术证明了其功能（初步数据）。我们的中心假设是TRPM 4代表了控制干细胞中Ca 2+进入的关键调节机制，并且是增强组织再生的潜在靶点。该实验将确定TRPM 4在牙囊干细胞中的作用。在目标#1中，我们将确定TRPM 4是否介导干细胞分化为骨和脂肪。将在成骨细胞或脂肪细胞分化培养基中培养稳定的TRPM 4敲除克隆，并使用茜素红S（成骨细胞分化）检查碱性磷酸酶活性和细胞外基质中Ca 2+沉积的存在，或使用油红O检查细胞内囊泡内的脂质形成（脂肪细胞分化）。我们还将使用RT-PCR和微阵列分析确定如果TRPM 4被抑制，成骨或成脂基因是否更快地打开/关闭。在目标#2中，我们将确定TRPM 4是否控制Ca 2+信号。首先，我们将使用膜片钳技术对TRPM 4进行详细的定量分析和表征。第二，我们将检验抑制TRPM 4将增强干细胞中Ca 2+进入的假设。我们将用两种方法敲除TRPM 4：1）使用shRNA（稳定系统），和2）使用显性负性构建体（瞬时系统）。我们将使用实时数字成像分析评估TRPM 4敲低对G蛋白偶联受体激动剂和自发振荡产生的Ca 2+信号的影响。 公共卫生相关性：干细胞治疗为颅面缺损的修复提供了一种先进可靠的治疗策略。然而，决定干细胞分化的基本过程还没有得到很好的理解。这项研究的总体目标是了解钙信号如何影响干细胞的命运（例如分化为骨骼，脂肪，肌肉等）。离子通道控制钙信号的形状和频率是公认的。根据钙的模式，不同的基因可以被打开或关闭。事实上，钙信号的物理操纵促进干细胞分化成骨。钙信号还可以激活转录因子，使干细胞成为脂肪组织。因此，如果我们能够在钙离子模式和干细胞命运之间建立联系，就有可能通过靶向离子通道来产生专门的组织。这可能为探索组织工程或患者移植干细胞的独特性质提供了另一种方法。主要研究者在同行评审的科学期刊上发表了17篇手稿，其中包括4篇关于TRPM 4在免疫和胰腺细胞中调节钙信号的论文，目前正在将所获得的知识扩展到干细胞生物学。

DESCRIPTION (provided by applicant): Dysfunction or defects of craniofacial tissues has a significant impact on the patient's life. Therefore, reconstruction of damaged tissues is highly desired. Dental follicle stem cells are a potential source for tissue replacement due to their ability to differentiate into specialized cells, including bone and fat (1). However, fundamental processes in determining the differentiation pathways remain undetermined. Thus, our long-term goal is to characterize the molecular mechanisms controlling stem cell differentiation so that more rigorous and reliable protocols can be established for reconstruction of craniofacial components. Calcium (Ca2+) oscillations are a phenomenon commonly observed during stem cell differentiation and there is evidence that it may be important for directing and terminating the process (2-4). The Transient Receptor Potential Melastatin 4 (TRPM4) channel is essential for Ca2+ oscillations because it controls the amount of Ca2+ entering cells (5). Molecular suppression of TRPM4 increases Ca2+ entry and cytokine production in immune cells (5). In fact, enhanced Ca2+ entry also facilitates the differentiation of stem cells into bone and fat (2, 3). We have identified TRPM4 in stem cells using a molecular approach and demonstrated its functionality with the patch-clamp technique (preliminary data). Our central hypothesis is that TRPM4 represents a key regulatory mechanism that controls Ca2+ entry in stem cells and is a potential target to enhance tissue regeneration. The proposed experiments will determine the role of TRPM4 in dental follicle stem cells. In Aim #1, we will determine whether TRPM4 mediates stem cell differentiation into bone and fat. Stable TRPM4 knockdown clones will be cultured in osteoblast or adipocyte differentiation medium and examined for alkaline phosphatase enzyme activity and for the presence of Ca2+ deposition in the extracellular matrix using Alizarin Red S (osteoblast differentiation) or lipid formation inside intracellular vesicles using Oil Red O (adipocyte differentiation). We will also determine if osteogenic or adipogenic genes are more rapidly turned on/off if TRPM4 is inhibited using RT-PCR and microarray analysis. In Aim #2, we will determine whether TRPM4 controls Ca2+ signals. First, we will perform a detailed quantitative analysis and characterization of TRPM4 using the patch-clamp technique. Second, we will test the hypothesis that inhibition of TRPM4 will enhance Ca2+ entry in stem cells. We will knockdown TRPM4 with two approaches: 1) Use of the shRNA (stable system), and 2) Use of Dominant Negative constructs (transient system). We will assess the impact of TRPM4 knockdown on Ca2+ signals generated by G-protein coupled receptor agonists and spontaneous oscillations using real-time digital imaging analysis. PUBLIC HEALTH RELEVANCE: Stem cell therapy offers a promising approach to providing an advanced and reliable therapeutic strategy to repair craniofacial defects. However, fundamental processes determining stem cell differentiation are not well understood. The overall goal of this research is to understand how calcium signals impact the fate of stem cells (e.g. differentiation into bone, fat, muscle and others). It is well established that ion channels control the shape and frequency of calcium signals. Depending on the calcium pattern, different genes can be turned on or off. In fact, physical manipulation of calcium signals facilitates stem cell differentiation into bone. Calcium signals can also activate transcription factors to make stem cells become fat tissue. Therefore, if we can establish a link between calcium patterns and stem cell fate, it would be possible to generate specialized tissues by targeting ion channels. This may offer an alternative approach to explore the unique properties of stem cells for tissues engineering or patient transplant. The principal investigator has published 17 manuscripts in peer-reviewed scientific journals, including 4 papers on the regulation of calcium signals by TRPM4 in immune and pancreatic ¿-cells and is currently expanding the knowledge obtained to stem cell biology.