Inhibited Intramembranous Bone Healing in Diabetes

描述（由申请人提供）：糖尿病性骨病是一种骨骼变弱的疾病，导致活动能力降低，足部和其他骨折风险增加，牙周疾病更严重，生活质量普遍下降。已知这种情况主要发生在I型糖尿病患者中，尽管现在认为II型糖尿病患者也会发生骨质不良。糖尿病性骨疾病的特征是成骨细胞或骨合成活性降低。晚期糖基化终末产物（AGEs）在糖尿病患者骨中高水平积累，并由非酶促反应引起。TNF-α是在糖尿病的矿化和非矿化组织中以升高水平存在的细胞因子。AGEs和TNF-α各自导致糖尿病并发症，并且各自与成骨细胞表面受体相互作用，导致活性氧（ROS）水平增加。增加的ROS刺激FOXO 1的合成和核定位，FOXO 1是一种导致细胞周期停滞和细胞凋亡增加的转录因子。FOXO 1可以结合到由经典Wnt途径调节的转录辅因子-连环蛋白。Wnt通路促进成骨细胞增殖和分化。这里要检验的主要假设是AGEs和TNF-α各自刺激活性核FOXO 1水平的增加，FOXO 1与TCF/LEF转录因子竞争可用的<$-连环蛋白库。这被认为有效地抑制Wnt刺激的成骨细胞分化，并有助于糖尿病骨质减少。初步数据支持细胞外基质酶赖氨酰氧化酶的缺乏导致类似于骨质减少性糖尿病骨的不良骨结构，并且赖氨酰氧化酶通过成骨细胞中的Wnt经典途径上调。提出了两个具体目标。目的1将在体外和体内研究AGEs和TNF-α各自通过竞争有限的连环蛋白库来抑制经典Wnt通路的假设。目的2：探讨Wnt对赖氨酰氧化酶的调控机制，以及AGE和TNF-α抑制Wnt刺激的赖氨酰氧化酶产生的机制。目的2将进一步确定糖尿病小鼠颅骨缺损中经典Wnt通路和LOX表达的下调程度以及FOXO依赖性基因表达的上调程度。体内研究将利用在非糖尿病和糖尿病转基因小鼠中产生的颅骨缺陷的分析，所述转基因小鼠表达驱动半乳糖苷酶表达的典型Wnt途径应答启动子（TOPGAL小鼠）。在TOPGAL报告小鼠模型中产生的颅盖缺陷与糖尿病诱导的这种独特组合将提供作为糖尿病的函数以及作为AGE和TNF-α治疗的函数的经典Wnt途径活性的新的直接分析。将使用MC 3 T3成骨细胞和原代颅骨成骨细胞进行体外研究。研究结果将提供最终可能有助于预防或逆转糖尿病对骨质量影响的信息。公共卫生相关性：骨质减少或骨质疏松是糖尿病的并发症。晚期糖基化终产物（AGE）和细胞因子TNF-α在糖尿病组织中升高，并导致糖尿病的许多并发症。本研究的假设是AGE和TNF-α均抑制成骨细胞（骨合成细胞）中的Wnt经典途径，从而抑制成骨细胞的分化和发育以及赖氨酰氧化酶（骨形成所需的重要细胞外基质酶）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.