Role of Gsalpha in Regulating Osteoblast Differentiation

骨质疏松症是一种极为常见的退行性疾病，由骨形成和骨吸收失衡引起。这种差异随着年龄的增长而加剧，大约50%的50岁以上的女性会经历骨质疏松性骨折。引导骨形成细胞或成骨细胞分化的分子机制尚不清楚。Gsa是一种普遍表达的G蛋白亚基，介导多种G蛋白偶联受体（gpcr）下游的信号级联反应。几种gpcr与骨骼发育有关，特别是甲状旁腺激素(PTH)/PTH相关肽受体（PPR）在成骨细胞发育中具有关键功能。我们假设Gsa可能在骨发育的多个阶段具有重要功能，并且在成骨细胞谱系的早期Gsa的消融将对成骨细胞分化产生深远的影响。通过将Gsafloxed小鼠与携带由osterix启动子驱动的Cre重组酶的转基因小鼠配对，我们有条件地删除了早期成骨细胞前体中的Gsa， osterix是成骨细胞早期表达的转录因子。在成骨细胞分化早期缺失Gsa信号的小鼠具有明显的骨骼脆弱性，如许多出生后骨折和严重减少的小梁骨和皮质骨。此外，骨钙素（终末分化成骨细胞的标志）的表达几乎不存在。破骨细胞的TRAP染色并没有显示骨吸收的急剧增加，这使得骨形成失败更可能是这些突变小鼠骨量减少的病因。这项拨款提案的重点将是Gsa缺乏对成骨细胞分化的影响。绿色荧光蛋白（GFP）在这些转基因小鼠中的表达受骨脂调节，进一步提供了机会来确定通常用于成骨细胞分化的体外测定方法如何以及是否与体内实际事件相对应。目的1旨在确定Gsa如何调节成骨细胞分化；这将在体内通过原位杂交进行评估，并在体外通过在成骨条件下培养野生型和BGsaKO小鼠的骨髓基质细胞进行评估。此外，流式细胞术和荧光活化细胞分选（FACS）将用于分离成骨细胞谱系的细胞，以确定Gsa用于指导成骨细胞分化的机制。PTH和典型Wnt信号通路均可显著刺激骨形成，Aim 2旨在确定gsa介导的信号通路的缺失是否会导致Wnt信号通路的抑制，并以此作为BGsaKO小鼠骨量严重减少的机制。最后，Aim 3将侧重于确定骨祖细胞向其他间充质细胞（如脂肪细胞）分化的能力，使用体内谱系追踪和体外细胞分化试验。

DESCRIPTION (provided by applicant): Osteoporosis is an extremely common degenerative disease resulting from an imbalance between bone formation and bone resorption. This discrepancy worsens with age, such that approximately 50% of women over the age of 50 will experience an osteoporotic fracture. The molecular mechanisms guiding the differentiation of bone forming cells, or osteoblasts, are not clearly understood. Gsa is a ubiquitously expressed G protein subunit that mediates signaling cascades downstream of a variety of G protein-coupled receptors (GPCRs). Several GPCRs have been implicated in skeletal development, and in particular the parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) has a critical function in osteoblast development. We have hypothesized that Gsa likely has essential functions at multiple stages of bone development and that ablation of Gsa early in the osteoblastic lineage will have profound effects on osteoblast differentiation. We have conditionally deleted Gsa in early osteoblast precursors by mating Gsafloxed mice with transgenic mice carrying the Cre recombinase driven by the promoter of osterix, a transcription factor expressed early in osteoblastogenesis. Mice with deletion of Gsa signaling early in osteoblast differentiation have marked skeletal fragility as demonstrated by numerous postnatal fractures and severely reduced trabecular and cortical bone. Furthermore expression of osteocalcin, a marker of terminally differentiated osteoblasts, is almost absent. TRAP staining for osteoclasts does not reveal dramatic increase in bone resorption, making a failure of bone formation the more likely etiology for reduced bone mass in these mutant mice. The focus of this grant proposal will be on the impact of Gsa deficiency on osteoblast differentiation. The expression of green fluorescent protein (GFP) regulated by osterix in these genetically altered mice further provides the opportunity to determine how and whether commonly used in vitro assays for osteoblast differentiation correspond to actual events in vivo. Aim I seeks to determine how Gsa regulates osteoblast differentiation; this will be assessed in vivo using in situ hybridization, and in vitro by culturing bone marrow stromal cells from wild-type and BGsaKO mice under osteogenic conditions. Furthermore, flow cytbmetry and fluorescence-activated cell sorting (FACS) will be applied to isolate cells of the osteoblast lineage in order to determine the mechanisms used by Gsa to direct osteoblast differentiation. Bone formation is dramatically stimulated by both PTH and canonical Wnt signaling pathways, and Aim 2 seeks to determine whether the loss of Gsa-mediated signaling results in inhibition of Wnt signaling as a mechanism underlying the profoundly reduced bone mass in BGsaKO mice. Finally, Aim 3 will focus on determining the ability of osteoprogenitors to differentiate towards other mesenchymal lineages such as adipocytes, using lineage tracing in vivo and cell differentiation assays in vitro.