Osteocytes in osteogenesis imperfecta (OI)

项目摘要/摘要--项目2 成骨不全(OI)通常由I型胶原基因突变引起，更罕见的是由 I型胶原蛋白加工相关基因的隐性突变(例如，CRTAP)。因为I型胶原蛋白是 成骨细胞分泌的最丰富的基质成分，这些细胞一直被认为 疾病表现的主要介体。因此，骨细胞在骨骼中可能扮演的角色 OI的表现在很大程度上被忽视了。因此，项目2的总体目标是阐明 骨细胞在OI骨脆性中的作用并确定骨细胞转录组的变化可能 引导出新的治疗靶点。我们的初步数据显示，骨细胞表达的I型胶原与 成骨细胞，提示I型胶原的改变，除了影响成骨细胞外，也可能是负面的。 冲击骨细胞。为了支持这一点，我们发现来自2个OI小鼠模型的骨细胞转录本 与对照小鼠相比，它们的基因表达显著失调。此外，骨细胞积极表达所有19个基因。 与OI的显性和隐性形式有关。然而，目前尚不清楚胶原蛋白在多大程度上发生了变化 OI影响骨细胞的功能，以及它们的转录失调是否是 由骨细胞表达的改变的胶原蛋白，或由于它们与骨细胞产生的改变的胶原蛋白相互作用而产生的 成骨细胞。我们的中心假设是骨细胞导致了OI中观察到的骨脆性，并且 骨细胞中I型胶原的表达改变会破坏骨细胞的功能，进而改变骨细胞 调节骨骼动态平衡的基因表达。为了验证这一假设，我们生成了一个新的敲入 Cre介导的重组可用于表达OI甘氨酸取代的小鼠模型 内源性Col1a1基因(p.Gly1146Arg)突变。我们将用EIIA-CRE和SOST-CRE培育这只小鼠 小鼠，全局或特异性地在骨细胞中表达突变，然后比较 使用双X射线吸收测量、微型CT、骨生物力学、骨组织形态计量学和Q-PCR的后代 评估在骨细胞特异性敲入OIOCY小鼠中复制OI骨骼表型的哪些方面 (目标1)。我们还将为胶原修饰基因(CRTAP)生成条件等位基因，并确定 在骨细胞中缺失它的骨骼后果与使用所述方法学的全局失活相比 以上(目标2)。为了确定OI中的骨细胞功能是否因为表达异常的胶原而受到影响， 因为它们与成骨细胞分泌的有缺陷的骨基质相互作用，或者两者兼而有之，我们将识别骨细胞 骨细胞特异性CRTAP KO、全局CRTAP KO和WT小鼠之间的转录组差异 和单细胞RNA-seq(目标3)。这些研究将提供改变程度的直接证据 胶原蛋白影响骨细胞功能导致OI引起的部分骨缺陷及鉴定 骨细胞的转录变化可能为治疗干预提供新的靶点。

PROJECT SUMMARY/ABSTRACT – PROJECT 2 Osteogenesis imperfecta (OI) is usually caused by mutations in type I collagen genes and more rarely, by recessive mutations in genes involved in type I collagen processing (e.g., CRTAP). Because type I collagen is the most abundant matrix component secreted by bone forming osteoblasts, these cells have been considered the primary mediators of the disease presentation. Consequently, a possible role for osteocytes in the skeletal manifestations of OI has largely been overlooked. Thus, the overall goal of Project 2 is to elucidate the contribution of osteocytes to bone fragility in OI and identify changes in the osteocytic transcriptome that may lead to new therapeutic targets. Our preliminary data show that osteocytes express as much type I collagen as osteoblasts, suggesting that alterations in type I collagen, in addition to affecting osteoblasts, may also negatively impact osteocytes. In support of this, we found that the osteocyte transcriptomes from 2 mouse models of OI were significantly dysregulated compared to control mice. Moreover, osteocytes actively express all 19 genes associated with dominant and recessive forms of OI. However, it is unknown to what extent altered collagen in OI affects the function of osteocytes, and whether their transcriptional dysregulation is a direct consequence of the altered collagen expressed by osteocytes or due to their interaction with altered collagen produced by osteoblasts. Our central hypothesis is that osteocytes contribute to the bone fragility observed in OI and that expression of altered type I collagen in osteocytes disrupts osteocyte function and, in turn, alters the osteocytic expression of genes that regulate bone homeostasis. To test this hypothesis, we generated a novel knock-in mouse model in which Cre-mediated recombination can be used to express an OI glycine substitution (p.Gly1146Arg) mutation in the endogenous Col1a1 gene. We will breed this mouse with EIIa-Cre and Sost-Cre mice, to express the mutation globally or specifically in osteocytes, and then compare the skeletal phenotype of offspring using dual X-ray absorptiometry, micro-CT, bone biomechanics, bone histomorphometry, and Q-PCR to assess what aspects of the OI skeletal phenotype are reproduced in osteocyte-specific knock-in OIOCY mice (Aim 1). We will also generate a conditional allele for a collagen-modifying gene (Crtap) and determine the skeletal consequences of its deletion in osteocytes versus global inactivation using the methodologies described above (Aim 2). To determine if osteocyte function in OI is affected because they express abnormal collagen, because they interact with a defective bone matrix secreted by osteoblasts, or both, we will identify osteocyte transcriptome differences between osteocyte-specific Crtap KO, global Crtap KO, and WT mice using both bulk and single-cell RNA-seq (Aim 3). These studies will provide direct evidence of the extent to which altered collagen affects osteocyte function leading to some of the skeletal defects caused by OI and identify transcriptional changes in osteocytes that may provide new targets for therapeutic interventions.