Role of TRPM4 in dental follicle stem cell differentiation

描述（由申请人提供）：颅面组织功能障碍或缺陷对患者的生活有重大影响。因此，受损组织的重建是非常需要的。牙滤泡干细胞是组织替代的潜在来源，因为它们能够分化为特化细胞，包括骨和脂肪(1)。然而，决定分化途径的基本过程仍未确定。因此，我们的长期目标是描述控制干细胞分化的分子机制，以便为颅面成分的重建建立更严格和可靠的方案。钙（Ca2+）振荡是干细胞分化过程中常见的一种现象，有证据表明它可能对指导和终止这一过程很重要（2-4）。瞬时受体电位美拉他汀4 （TRPM4）通道对Ca2+振荡至关重要，因为它控制Ca2+进入细胞的数量(5)。分子抑制TRPM4增加免疫细胞中Ca2+的进入和细胞因子的产生(5)。事实上，增强的Ca2+进入也促进了干细胞向骨和脂肪的分化（2,3）。我们使用分子方法鉴定了干细胞中的TRPM4，并通过膜片钳技术证明了其功能（初步数据）。我们的中心假设是TRPM4代表了控制Ca2+进入干细胞的关键调节机制，是增强组织再生的潜在靶点。本实验将确定TRPM4在牙滤泡干细胞中的作用。在Aim #1中，我们将确定TRPM4是否介导干细胞向骨和脂肪的分化。稳定的TRPM4敲低克隆将在成骨细胞或脂肪细胞分化培养基中培养，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Ca2+沉积的存在，或使用油红O（脂肪细胞分化）检查细胞内囊泡内脂质形成。我们还将通过RT-PCR和微阵列分析确定，如果TRPM4被抑制，成骨或脂肪基因是否会更快地开启/关闭。在Aim #2中，我们将确定TRPM4是否控制Ca2+信号。首先，我们将使用膜片钳技术对TRPM4进行详细的定量分析和表征。其次，我们将验证抑制TRPM4会增强干细胞中Ca2+进入的假设。我们将通过两种方法敲除TRPM4: 1)使用shRNA（稳定系统）和2)使用显性负性结构（瞬时系统）。我们将利用实时数字成像分析评估TRPM4敲低对g蛋白偶联受体激动剂和自发振荡产生的Ca2+信号的影响。

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.