Transgenic approaches to evaluate IGF-I role in peak BMD

描述（由申请人提供）：骨质疏松症是一种以低峰值骨量为特征的疾病，导致骨折在最小创伤后发生时脆性增加。因此，在骨质疏松症的预防和治疗中相当重要的是那些集中于理解在发生快速骨增长的关键窗口期间调节骨增长的机制的研究。为此，我们的研究涉及条件性敲除IGF-I的Cre/LoxP的方法在过去的资助期间提供了明确的实验证据，IGF-I在骨局部产生的青春期前生长期的峰值骨量的收购中起着关键作用。我们的研究还得出了一个意想不到的重要结论，即青春期前生长期间大量的IGF-I作用是由生长激素（GH）非依赖性机制调节的。在这个继续资助，我们提出了一个新的假设，即在青春期前生长期的快速增加IGF-I表达的调节甲状腺激素（TH），独立的GH。为了检验这一假设，即IGF-I的表达在青春期前的生长期主要是由TH调节，我们将使用TSH受体和双氧化酶2突变的小鼠模型，这是缺乏TH和测试的原因和影响之间的关系增加TH生产和IGF-I的表达在青春期前的生长期。为了确定TH调节IGF-I表达独立于GH，我们将使用GH释放激素受体突变的GH缺陷lit/lit小鼠模型。为了验证这一假设，即TH对IGF-I表达的影响在青春期前的生长期是通过TH受体（TR）α 1介导的，我们将检查TRa 1和IGF-I是否在骨中的特定细胞类型中共表达。我们将通过条件性基因敲除的方法破坏成骨细胞中的TRa 1功能，并评估TRa 1破坏对青春期前生长期IGF-I表达和骨骼表型的影响。为了验证TH对骨的作用是通过增加骨细胞产生IGF-I来介导的这一假设，我们将使用腺病毒Cre从floxed成骨细胞中删除IGF-I，并在体外测试TH对成骨细胞增殖和分化的作用。为了测试局部产生的IGF-I对体内骨形成的作用，我们将测试TH对条件性IGF-I敲除小鼠（其中IGF-I基因在成骨细胞中被破坏）和相应对照小鼠中骨骼变化的影响。由于IGF-I是一种非常重要的生长因子，并且由于对青春期前生长期间IGF-I表达的调节知之甚少，因此我们假设TH是青春期前生长期间IGF-I表达的主要调节剂，并且TH对骨的作用是通过局部产生IGF-I介导的，这一假设的证实可能导致基于TH作用的有效疗法的开发，在快速生长的窗口期治疗患有生长障碍的儿童。 公共卫生相关性：在儿童早期形成的骨量是一个非常重要的决定因素，就个体是否会在以后的生活中患上骨质疏松症而言。因此，了解早期生长期骨骼变化的分子机制是非常重要的。本研究将检验一种新的假设，即胰岛素样生长因子（IGF）-I对骨骼生长的影响主要是通过甲状腺激素介导的，其机制独立于生长激素。由于IGF-I被证明是一个非常重要的分子，在调节骨形成过程中，IGF-I的假设，在响应甲状腺激素在骨局部产生发挥了重要作用，在骨增生的快速增加，发生在青春期前生长期的独特的时间窗口，将有未来的分歧有关的策略，以增加峰值骨量。

DESCRIPTION (provided by applicant): Osteoporosis is a disease characterized by low peak bone mass leading to higher fragility where fractures occur after minimal trauma. Therefore, of considerable importance in the prevention and treatment of osteoporosis are those studies that focus on understanding the mechanisms regulating bone accretion during critical windows in time when rapid bone accretion takes place. To this end, our studies involving conditional knockout of IGF-I by Cre/LoxP approach during the past grant period have provided unequivocal experimental evidence that IGF-I produced locally in bone plays a key role in the acquisition of peak bone mass during prepubertal growth period. Our studies also have lead to an unexpected important conclusion that a great deal of the IGF-I action during the prepubertal growth period is regulated by a growth hormone (GH)-independent mechanism. In this continuation grant, we have proposed a novel hypothesis that the rapid increase in IGF-I expression during prepubertal growth period is regulated by thyroid hormone (TH), independent of GH. To test the hypothesis that IGF-I expression during prepubertal growth period is regulated predominantly by TH, we will use TSH receptor and dual oxidase 2 mutant mouse models which are deficient in TH and test the cause and effect relationship between an increase in TH production and IGF-I expression during the prepubertal growth period. To establish that TH regulates IGF-I expression independent of GH, we will use GH-deficient lit/lit mouse model with a mutation in GH-releasing hormone receptor. To test the hypothesis that the TH effect on IGF-I expression during the prepubertal growth period is mediated via TH receptor (TR)a1, we will examine if TRa1 and IGF-I are coexpressed in specific cell types in bone. We will disrupt TRa1 function in osteoblasts by conditional knockout approach and evaluate the consequence of TRa1 disruption on IGF-I expression and skeletal phenotype during prepubertal growth period. To test the hypothesis that TH effects on bone are mediated via increased bone cell production of IGF-I, we will delete IGF-I from floxed osteoblasts using adenoviral Cre and test TH effects on osteoblast proliferation and differentiation in vitro. To test the role of locally produced IGF-I on bone formation in vivo, we will test the effects of TH on skeletal changes in conditional IGF-I knockout out mice in which IGF-I gene is disrupted in osteoblasts and corresponding control mice. Because IGF-I is a very important growth factor and because little is known about the regulation of IGF-I expression during prepubertal growth period, the confirmation of our hypothesis that TH is a major regulator of IGF-I expression during prepubertal growth period and that TH effects on bone are mediated via local production of IGF-I, could lead to the development of effective therapies, based on TH action, to treat children with growth disorders during a window in time when rapid growth occurs. PUBLIC HEALTH RELEVANCE: The amount of bone formed early in childhood is a very important determining factor in terms of whether or not the individual will develop osteoporosis later in life. Therefore, it is great importance to understand the molecular mechanisms that contribute to skeletal changes that occur during the early growth period. This study will test a novel hypothesis that Insulin-like growth factor (IGF)-I effects on skeletal growth is predominantly mediated via thyroid hormone via a mechanism that is independent of growth hormone. Because IGF-I is proven to be an extremely important molecule in the regulation of bone formation process, the confirmation of the hypothesis that IGF-I produced locally in bone in response to thyroid hormone plays a major role in the rapid increase in bone accretion that takes place during the unique window in time during the prepubertal growth period will have future ramifications concerning strategies to increase peak bone mass.