TRANSGENIC EXPRESSION OF 11B-HSD2 IN BONE

描述(由申请人提供)：这项建议建立在我们目前的研究努力基础上，以了解糖皮质激素在骨发育和骨重塑中的作用，使用新的遗传策略。骨生物学中一个长期存在的悖论是，糖皮质激素在体内和体外对成骨的影响似乎是不同的。已发表的文献表明，在各种体外模型中，糖皮质激素可以促进成骨细胞分化和骨形成，尽管在人类和小鼠中，高水平的糖皮质激素通常会减少骨量并抑制骨形成标志物。体外观察到的糖皮质激素成骨作用的意义尚不清楚，尽管这可能反映了在维持成骨细胞表型方面的许可作用。我们最近培育了以成骨细胞为靶点表达11β-羟基类固醇脱氢酶-2(11β-HSD2)的转基因小鼠，这是一种代谢酶，通过将生物活性的糖皮质激素转化为非活性的11-酮代谢物来扰乱糖皮质激素信号转导。这一方法将使我们能够阻断骨骼中所有已知的糖皮质激素信号通路，并评估糖皮质激素在体内骨骼发育和重塑中的生理作用。到目前为止，11β-HSD2在成熟成骨细胞中的转基因表达导致了一种迷人的性别和骨骼部位相关的骨表型，其中雌性小鼠椎骨中的骨体积减少。该方案中的实验旨在扩展我们的初步研究，并验证内源性糖皮质激素是体内成骨细胞分化、成骨细胞功能和骨重建的关键调节因子的假说。为此，本研究的具体目标是：1)利用在成熟成骨细胞中表达11β-HSD2的Col2.3-HSD2转基因小鼠模型，确定内源性糖皮质激素影响成骨细胞功能和骨重建的细胞和分子机制；以及2)使用结构性活性和临时诱导的Col3.6-HSD2转基因模型，确定内源性糖皮质激素在成骨细胞分化和功能中的作用，在该模型中，11β-HSD2被更广泛地靶向成熟的成骨细胞及其祖细胞。

DESCRIPTION (provided by applicant): This proposal builds on our current research efforts to understand the role of glucocorticoids in bone development and remodeling using novel genetic strategies. A long-standing paradox in bone biology has been the seemingly disparate effects of glucocorticoids on osteogenesis in vivo and in vitro. Published literature shows that glucocorticoids can promote osteoblast differentiation and bone formation in a variety of in vitro models, even though high levels of glucocorticoids typically decrease bone mass and inhibit markers of bone formation in humans and mice. The significance of the osteogenic effect of glucocorticoids seen in vitro remains unclear, although this may reflect a permissive role in the maintenance of the osteoblast phenotype. We have recently generated transgenic mice with osteoblast-targeted expression of 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD2), a metabolic enzyme that disrupts glucocorticoid signaling by converting bioactive glucocorticoids to their inactive 11-keto-metabolites. This approach will allow us to block all known glucocorticoid signaling pathways in bone and assess the physiological role of glucocorticoids on bone development and remodeling in vivo. Thus far, transgenic expression of 11beta-HSD2 in mature osteoblasts results in a fascinating sex- and skeletal-site dependent bone phenotype in which there is reduction of bone volume in vertebrae of female mice. The experiments in this proposal are designed extend our preliminary studies and test the hypothesis that endogenous glucocorticoids are key regulators of osteoblast differentiation, osteoblast function and bone remodeling in vivo. To this end, the specific aims are: 1) To determine the cellular and molecular mechanisms by which endogenous glucocorticoids affect osteoblast function and bone remodeling using the Col2.3-HSD2 transgenic mouse model in which 11beta-HSD2 is expressed in mature osteoblasts; and 2) To determine the role of endogenous glucocorticoids in osteoblast differentiation and function using a constitutively active and temporally inducible Col3.6-HSD2 transgenic models in which 11beta-HSD2 is targeted more broadly to mature osteoblasts and their progenitors.