Vesicle Trafficking and Osteoblast Function

项目摘要 骨质疏松症是一种骨骼脆性疾病，50%的女性和25%的男性年龄以上会导致骨折 50美元。最常用的抗骨质疏松症药物无法治愈骨质疏松症，而 目前可用的骨形成合成代谢骨质疏松症药物因疗效减弱而受到限制。仍然有 对安全和持续的增加骨形成的方法的巨大需求尚未得到满足。我们已经展示了 2.3kb的大鼠I型胶原启动子驱动的绿色荧光蛋白(Col2.3GFP)在 成熟成骨细胞。我们从骨骼中分离出Col2.3GFP(Hi)成骨细胞，培养骨片，并定向 胚胎干细胞的分化，并通过RNA测序鉴定了593个成熟富含基因 成骨细胞。基因本体论(GO)分析发现，内质网到高尔基体小泡的运输是最丰富的 去学期吧。我们的初步数据显示，在MC3T3成骨细胞中，几个囊泡的瞬时敲除 转移基因导致矿化结节形成增加和成骨细胞标志物加速 表情。我们的中心假设是，囊泡运输的中断会损害骨形成，这是由于 胶原蛋白过度修饰和过度矿化。我们将利用几种创新方法：直接 对成纤维细胞重新编程以获得诱导成骨细胞，编辑CRISPR/Cas9基因以删除囊泡 诱导成骨细胞中的运输基因，作为细胞自主成骨细胞报告的Col2.3GFP，以及 基因编辑的成骨细胞皮下移植以评估体内骨形成。我们筛查了水泡 瞬时siRNA敲除运输基因，筛选出9个基因(Preb、Stx5a、Rab2a、Gosr2、Bet1、 RAMP1，Arf4，Cog6，Pacs1)，其基因敲除增加矿化结节形成，成骨细胞标志物 表达和内质网应激。在目标1中，我们将确定囊泡运输的中断是否会增加 由于胶原蛋白过度修饰而导致的矿化。我们将通过CRISPR/Cas9基因进行永久敲除 用Col2.3GFP成骨细胞报告基因编辑小鼠和人诱导成骨细胞中的每个基因 评估成骨细胞标志物的表达和矿化结节的形成。我们将测量胶原蛋白的产量 以及翻译后通过脯氨酸羟化修饰，并确定是否抑制胶原 过度修饰可恢复成骨细胞功能。在目标2中，我们将确定水泡是否破裂 运输会损害体内的骨形成。我们将通过皮下观察体内的骨形成。 基因编辑的小鼠和人的iOB的移植。我们将确定是否抑制胶原蛋白 过度修饰可以恢复体内的骨形成。这些目标的成功完成将识别新的基因 参与骨形成，作为治疗骨质疏松症的潜在治疗靶点，并将提供 对囊泡运输机制在成骨细胞功能中作用的机械洞察。

Project Summary Osteoporosis is a disease of skeletal fragility that causes fractures in 50% of women and 25% of men over age 50. The most commonly prescribed anti-resorptive osteoporosis medications cannot cure osteoporosis, while currently available bone-building anabolic osteoporosis medications are limited by waning efficacy. There is still a great unmet need for safe and sustained approaches to increasing bone formation. We have shown that 2.3-kb rat type I collagen promoter-driven green fluorescent protein (Col2.3GFP) is highly expressed in mature osteoblasts. We isolated Col2.3GFP(hi) osteoblasts from bones, cultured bone chips, and directed differentiation of embryonic stem cells, and by RNA-sequencing identified 593 genes that are enriched in mature osteoblasts. Gene ontology (GO) analysis identified ER-to-Golgi vesicle trafficking as the most highly enriched GO term. Our preliminary data reveal that transient knockdown in MC3T3 osteoblasts of several vesicle trafficking genes results in increased mineralized nodule formation and accelerated osteoblast marker expression. Our central hypothesis is that disruption of vesicle trafficking impairs bone formation due to collagen overmodification and hypermineralization. We will leverage several innovative methods: direct reprogramming of fibroblasts to derive induced osteoblasts, CRISPR/Cas9 gene editing to delete vesicle trafficking genes in induced osteoblasts, Col2.3GFP as a cell-autonomous osteoblast reporter, and subcutaneous transplantation of gene-edited osteoblasts to assess in vivo bone formation. We screened vesicle trafficking genes with transient siRNA knockdown, and selected 9 genes (Preb, Stx5a, Rab2a, Gosr2, Bet1, Ramp1, Arf4, Cog6, Pacs1) whose knockdown increased mineralized nodule formation, osteoblast marker expression, and ER stress. In Aim 1 we will determine whether disruption of vesicle trafficking increases mineralization due to collagen overmodification. We will perform permanent knockdown by CRISPR/Cas9 gene editing of each gene in mouse and human induced osteoblasts with the Col2.3GFP osteoblast reporter and assess osteoblast marker expression and mineralized nodule formation. We will measure collagen production and post-translational modification by prolyl hydroxylation, and determine whether inhibition of collagen overmodification can restore osteoblast function. In Aim 2 we will determine whether disruption of vesicle trafficking impairs bone formation in vivo. We will examine bone formation in vivo by subcutaneous transplantation of gene-edited mouse and human iOBs. We will determine whether inhibition of collagen overmodification restores bone formation in vivo. Successful completion of these aims will identify novel genes involved in osteogenesis as potential therapeutic targets for the treatment of osteoporosis, and will provide mechanistic insights into the role of vesicle trafficking machinery in osteoblast function.