Role of Gsalpha in Regulating Osteoblast Differentiation

描述(申请人提供)：骨质疏松症是一种非常常见的退行性疾病，由骨形成和骨吸收之间的失衡引起。这种差异随着年龄的增长而恶化，因此大约50%的50岁以上的女性将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是一种广泛表达的G蛋白亚基，参与多种G蛋白偶联受体(GPCRs)下游的信号传导。一些GPCRs与骨骼发育有关，尤其是甲状旁腺激素(PTH)/甲状旁腺激素相关多肽受体(PPR)在成骨细胞发育中起着关键作用。我们假设GSA可能在骨发育的多个阶段具有基本功能，在成骨细胞谱系早期去除GSA将对成骨细胞分化产生深远的影响。我们已经有条件地删除了早期成骨细胞前体中的GSA，方法是将GSaflox小鼠与携带由Osterix启动子驱动的Cre重组酶的转基因小鼠交配，Osterix是成骨细胞形成早期表达的转录因子。在成骨细胞分化早期GSA信号缺失的小鼠有明显的骨骼脆性，出生后大量骨折和严重的骨小梁和皮质骨减少就证明了这一点。此外，作为成骨细胞终末分化标志的骨钙素几乎不表达。破骨细胞的TRAP染色没有显示骨吸收的显著增加，这使得骨形成失败更有可能是这些突变小鼠骨量减少的原因。这项资助提案的重点将是GSA缺乏对成骨细胞分化的影响。由Osterix调控的绿色荧光蛋白(GFP)在这些基因改变的小鼠中的表达进一步提供了机会，以确定常用的体外成骨细胞分化检测方法如何以及是否符合体内的实际情况。目的研究GSA对成骨细胞分化的调节作用，通过原位杂交法和体外成骨条件下培养野生型和BGsaKO小鼠的骨髓基质细胞，对其进行体内评价。此外，将应用流式细胞术和荧光激活细胞分选(FACS)分离成骨细胞系的细胞，以确定GSA用于指导成骨细胞分化的机制。甲状旁腺激素和典型的Wnt信号通路都极大地刺激了骨形成，Aim 2试图确定GSA介导的信号通路的丢失是否导致Wnt信号的抑制，作为BGsaKO小鼠骨量显著减少的机制。最后，目标3将通过体内谱系追踪和体外细胞分化试验，重点确定成骨前体细胞向其他间充质细胞系分化的能力。

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.