Role of Gsalpha Signaling in Osteoblasts

描述（由申请人提供）：Gsa是一种普遍存在的G蛋白a亚基，其在刺激包括PTH/PTHrP受体（PPR）在内的多种受体时激活腺苷酸环化酶。Gsa的失活和激活突变都对骨骼发育有影响。为了确定Gsa信号转导在体内成骨细胞发育过程中的作用，我们通过将Gsa-floxed小鼠与osterix启动子-Cre重组酶转基因小鼠交配，有条件地删除了早期成骨细胞前体中的Gsa。具有早期成骨细胞特异性Gsa失活（BGsaKO）的小鼠具有显著的骨骼脆弱性，并且在出生后第1天表现出许多骨折。来自BGsaKO小鼠的长骨表现出小梁骨和皮质骨的严重减少。具体目的我将评估Gsa消融在成骨细胞前体中对成骨细胞分化、骨形成和骨吸收的生理学影响。在特定目标II中，重点将是从BGsaKO小鼠中分离骨髓基质细胞。然后，这些细胞可用于探测调节成骨细胞发育的信号通路，特别是检查Gsa是否是PTH对成骨细胞特异性基因表达的PKA依赖性刺激所必需的。

DESCRIPTION (provided by applicant): Gsa is a ubiquitous G protein a subunit which activates adenylyl cyclase upon stimulation of a variety of receptors including the PTH/PTHrP receptor (PPR). Both inactivating and activating mutations in Gsa have consequences in skeletal development. To determine the role of Gsa signaling during osteoblast development in vivo, we have conditionally deleted Gsa in early osteoblast precursors by mating Gsa-floxed mice with osterix promoter-Cre recombinase transgenic mice. Mice with early osteoblast-specific inactivation of Gsa (BGsaKO) have marked skeletal fragility and exhibit numerous fractures by postnatal day 1. Long bones from BGsaKO mice demonstrate severe reduction in trabecular and cortical bone. Specific Aim I will evaluate the physiologic effects of Gsa ablation in osteoblast precursors on osteoblast differentiation, bone formation and bone resorption in vivo. In Specific Aim II the focus will be to isolate bone marrow stromal cells from BGsaKO mice. These cells can then be used to probe the signaling pathways regulating osteoblast development, in particular to examine whether Gsa is required for the PKA-dependent stimulation of osteoblast-specific gene expression by PTH.