Inhibited Intramembranous Bone Healing in Diabetes

描述（由申请人提供）：糖尿病骨病是一种骨骼变弱的疾病，导致活动能力下降，足部和其他骨折的风险增加，更严重的牙周病，以及总体生活质量下降。这种情况已知主要发生在I型糖尿病患者中，尽管现在了解到骨质量差也发生在II型糖尿病患者中。糖尿病性骨病的特征是成骨细胞或骨合成活性降低。晚期糖基化终产物（AGEs）积累在高水平的糖尿病骨，结果由非酶的反应。TNF-a是一种细胞因子，在糖尿病的矿化和非矿化组织中水平升高。AGEs和TNF-a都与糖尿病并发症有关，它们都与成骨细胞表面受体相互作用，导致活性氧（ROS）水平升高。增加的ROS刺激fox01的合成和核定位，fox01是一种导致细胞周期阻滞和细胞凋亡增加的转录因子。fox01可以结合-catenin，一种由典型Wnt通路调节的转录辅助因子。Wnt通路促进成骨细胞增殖和分化。这里要测试的主要假设是，AGEs和TNF-a各自刺激活性核fox01水平的增加，fox01与TCF/LEF转录因子竞争可用的¿-连环蛋白库。这可能有效抑制Wnt刺激的成骨细胞分化，导致糖尿病骨质减少。初步数据支持细胞外基质酶赖氨酸氧化酶缺乏导致骨结构不良，类似于骨质减少的糖尿病骨，赖氨酸氧化酶在成骨细胞中通过Wnt规范途径上调。提出了两个具体目标。目的1将研究体外和体内的假设，即AGEs和TNF-a通过竞争有限的连环蛋白库来抑制典型的Wnt通路。目的2将确定Wnt调控赖氨酸氧化酶的机制，以及AGE's和TNF-a在体外抑制Wnt刺激的赖氨酸氧化酶生成的机制。Aim 2将进一步确定糖尿病小鼠颅骨缺陷中典型Wnt通路和LOX表达下调的程度，FOXO依赖基因表达上调的程度。体内研究将利用对非糖尿病和糖尿病转基因小鼠颅骨缺陷的分析，这些小鼠表达一个典型的Wnt通路响应启动子，该启动子驱动-半乳糖苷酶（TOPGAL小鼠）的表达。这种在TOPGAL报告小鼠模型中形成的颅部缺陷与糖尿病诱导的独特结合，将为典型Wnt通路活性作为糖尿病的功能，以及作为AGE和TNF-a治疗的功能提供一种新的直接分析。体外研究将使用MC3T3成骨细胞和原代颅骨成骨细胞进行。结果将为预防或逆转糖尿病对骨质量的影响提供最终有用的信息。公共卫生相关性：骨质减少或骨质疏松是糖尿病的一种并发症。晚期糖基化终产物（AGE's）和细胞因子TNF-a在糖尿病组织中升高，并导致糖尿病的许多并发症。本研究提出的假设是AGE和TNF-a分别抑制成骨细胞（骨合成细胞）中的Wnt规范通路，从而抑制成骨细胞的分化和发育，以及骨形成所需的重要细胞外基质酶赖氨酸氧化酶的合成。

DESCRIPTION (provided by applicant): Diabetic bone disease is a condition in which bones become weak, resulting in diminished mobility, increased risk of foot and other fractures, more severe periodontal diseases, and a generally diminished quality of life. This condition is known to occur mainly in Type I diabetics, although poor bone quality is now understood to occur in Type II diabetics as well. Diabetic bone disease is characterized by diminished osteoblastic or bone synthetic activity. Advanced glycation endproducts (AGEs) accumulate at high levels in diabetic bone, and result from non-enzymatic reactions. TNF-a is a cytokine present at elevated levels in mineralized and non-mineralized tissues in diabetes. AGEs and TNF-a each contribute to complications of diabetes, and each interacts with osteoblast cell surface receptors resulting in increased levels of reactive oxygen species (ROS). Increased ROS stimulates synthesis and nuclear localization of FOXO1, a transcription factor that leads to cell cycle arrest and increased apoptosis. FOXO1 can bind to ¿-catenin, a transcription cofactor that is regulated by the canonical Wnt pathway. The Wnt pathway promotes osteoblast proliferation and differentiation. The major hypothesis to be tested here is that AGEs and TNF-a each stimulate increased levels of active nuclear FOXO1, which competes with TCF/LEF transcription factors for the available pool of ¿-catenin. This is proposed to effectively inhibit Wnt stimulated osteoblast differentiation, and contribute to diabetic osteopenia. Preliminary data supports that deficiency of the extracellular matrix enzyme lysyl oxidase results in poor bone structure that resembles osteopenic diabetic bone, and that lysyl oxidase is up-regulated by the Wnt canonical pathway in osteoblasts. Two specific aims are proposed. Aim 1 will investigate in vitro and in vivo the hypothesis that AGEs and TNF-a each inhibit the canonical Wnt pathway by competing for a limited ¿-catenin pool. Aim 2 will determine the mechanism of Wnt regulation of lysyl oxidase, and the mechanism by which AGE's and TNF-a inhibit Wnt-stimulated lysyl oxidase production in vitro. Aim 2 will further determine in calvaria defects of diabetic mice the degree of down-regulation of the canonical Wnt pathway and LOX expression, and up-regulation of FOXO dependent gene expressions. In vivo studies will utilize analyses of calvaria defects made in non-diabetic and diabetic transgenic mice that express a canonical Wnt pathway responsive promoter that drives the expression of ¿-galactosidase (TOPGAL mouse). This unique combination of calvaria defects made in the TOPGAL reporter mouse model with diabetes induction will provide a novel direct analysis of canonical Wnt pathway activity as a function of diabetes, and as a function of AGE and TNF-a treatments. In vitro studies will be performed using MC3T3 osteoblasts and primary calvaria osteoblasts. Results will provide information that may be ultimately be useful in preventing or reversing effects of diabetes on bone quality. PUBLIC HEALTH RELEVANCE: Osteopenia, or weak bones, occurs as a complication of diabetes. Advanced glycation endproducts (AGE's) and the cytokine TNF-a are elevated in diabetic tissues, and contribute to many of the complications of diabetes. The proposed research investigates the hypothesis that AGE's and TNF-a each inhibit the Wnt canonical pathway in osteoblasts (bone synthetic cells), and thereby inhibit differentiation and development of osteoblasts, and synthesis of lysyl oxidase, an important extracellular matrix enzyme needed for bone formation.