Inhibited Intramembranous Bone Healing in Diabetes

描述(申请人提供)：糖尿病骨疾病是一种骨骼变得虚弱，导致活动能力下降，增加足部和其他骨折的风险，更严重的牙周疾病，以及普遍降低的生活质量的情况。已知这种情况主要发生在I型糖尿病患者中，尽管骨质量差现在被认为也发生在II型糖尿病患者中。糖尿病骨疾病的特征是成骨细胞或骨合成活动减少。晚期糖基化终末产物(AGEs)在糖尿病骨骼中大量积累，是非酶反应的结果。肿瘤坏死因子-α是一种细胞因子，在糖尿病矿化和非矿化组织中水平升高。糖化终末产物和肿瘤坏死因子-α均可导致糖尿病并发症，并与成骨细胞表面受体相互作用，导致活性氧簇(ROS)水平升高。增加的ROS刺激FOXO1的合成和核定位，FOXO1是一种导致细胞周期停滞和增加细胞凋亡的转录因子。Foxo1可以与转录辅因子？-catenin结合，该转录辅因子受典型的Wnt途径调节。Wnt途径促进成骨细胞的增殖和分化。这里要检验的主要假设是，AGEs和TNF-a各自刺激活跃的核FOXO1水平增加，FOXO1与TCF/Lef转录因子竞争可用的连接素池。这被认为可以有效地抑制Wnt刺激的成骨细胞分化，并有助于糖尿病骨量减少。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疏松性糖尿病骨的不良骨结构，并且赖氨酸氧化酶在成骨细胞中受到Wnt正则途径的上调。提出了两个具体目标。目的1将在体外和体内研究AGEs和TNF-a各自通过竞争有限的连环素池来抑制典型的Wnt途径的假设。目的2确定Wnt对赖氨酸氧化酶的调节机制，以及AGE和TNF-a体外抑制Wnt刺激的赖氨酸氧化酶产生的机制。目的2进一步确定糖尿病小鼠颅骨缺损区典型的Wnt通路和LOX表达下调的程度，以及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头盖骨缺陷的分析，这些转基因小鼠表达典型的Wnt途径反应启动子，驱动半乳糖苷酶(TOPGAL小鼠)的表达。这种在TOPGAL报告鼠模型中制造的颅骨缺陷与糖尿病诱导的独特组合，将提供一种新的直接分析经典Wnt途径活性作为糖尿病函数以及作为年龄和肿瘤坏死因子-a治疗函数的方法。体外研究将使用MC3T3成骨细胞和原代颅骨成骨细胞。研究结果将为预防或逆转糖尿病对骨骼质量的影响提供可能最终有用的信息。

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.