Targeting defective NO/cGMP signaling as novel therapy for diabetic osteoporosis

针对缺陷的 NO/cGMP 信号作为糖尿病骨质疏松症的新疗法

基本信息

批准号：
9899734
负责人：
RENATE B PILZ
金额：
$ 34.1万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9899734
关键词：
Affect Antioxidants Apoptotic Architecture Attention Biomechanics Bone Density Bone Diseases Calcium Cells Clinical Trials Complications of Diabetes Mellitus Cyclic GMP Cyclic GMP-Dependent Protein Kinases Data Defect Defense Mechanisms Development Diabetes Mellitus Enzymes Foundations Fracture Fracture Healing Gene Expression Gene Expression Profiling Generations Genes Glucose Glycogen Synthase Kinase 3 Growth Guanylate Cyclase Heme Group Hip Fractures Human Hydrogen Peroxide Impairment Insulin Insulin-Dependent Diabetes Mellitus Insulin-Like Growth Factor I Knockout Mice Lead Mechanical Stimulation Mediating Metabolism Mitochondria Molecular Mus NADPH Oxidase NOS3 gene Nitric Oxide Nitric Oxide Donors Nitric Oxide Synthase Non-Insulin-Dependent Diabetes Mellitus Osteoblasts Osteogenesis Osteoporosis Oxidative Stress Oxides Parents Pathway interactions Patients Pharmaceutical Preparations Phenotype Phosphorylation Play Post-Translational Protein Processing Postmenopause Production Reactive Oxygen Species Risk Role Secondary to Serum Markers Signal Pathway Signal Transduction Soluble Guanylate Cyclase Streptozocin Superoxides TXN gene Testing Tissues Transcriptional Regulation Viral Vitamin B 12 Vitamin D Wild Type Mouse Woman analog beta catenin bone bone loss bone quality cGMP production cobinamide diabetic diabetic patient effective therapy enzyme pathway fracture risk functional restoration gene repression improved in vivo microCT mouse model novel novel therapeutics osteoblast proliferation oxidation oxidative damage prevent protective effect public health relevance reconstitution response restoration skeletal substantia spongiosa treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Bone loss in diabetes has received surprisingly little attention, particularly considering that osteoporosis affects ~20% of patients with type 1 diabetes (T1DM) and poor bone quality is also a problem in type 2 diabetes. Bone loss is due to reduced osteoblastic bone formation, not increased osteoclastic resorption, thus, anti- resorptive therapies are inadequate. The molecular mechanisms of defective osteoblast function(s) are not well defined, but may include excessive production of reactive oxygen species (ROS). Nitric oxide (NO) plays an important role in osteoblasts, and clinical trials suggest that NO donors improve bone mineral density in post- menopausal women; however, conventional NO donors enhance oxidative stress. We previously showed that the NO/cGMP/protein kinase G (PKG) signaling pathway regulates osteoblast proliferation and survival. We found increased NADPH oxidase (NOX4) expression, excess ROS generation, decreased NO/cGMP production, and reduced PKG expression in bones and osteoblasts from mice with streptozotocin-induced T1DM, compared to control mice. Treatment with the cGMP-elevating agent cinaciguat, which is active even under high oxidative stress, largely restored defective proliferation and differentiation in diabetic (pre)osteo- blasts, and improved bone formation and trabecular bone volume in mice with T1DM. We hypothesize that defective NO/cGMP/PKG signaling-secondary in part to oxidative damage of pathway enzymes-contributes to bone loss in diabetes; restoring NO/cGMP signaling and/or reducing oxidative stress could be effective treatment(s) for diabetes-associated osteoporosis. The Specific Aims are to: (i) determine mechanisms and consequences of impaired NO/cGMP/PKG signaling in diabetic (pre)osteoblasts; (ii) analyze effects of cGMP- elevating agents on diabetes-induced bone loss in vivo; and (iii) examine the role of PKG and NOX4 in diabetes-induced bone loss. We will study the effects of diabetes on NO synthase and guanylate cyclase oxidation and post-translational modifications, and on PKG transcriptional regulation in osteoblasts. We will compare several approaches to prevent bone loss in mice with T1DM by treating the mice with: (i) cinaciguat; (ii) cobinamide, a vitamin B12 analog and potent anti-oxidant; (iii) the novel NO donor nitrosyl-cobinamide, which generates cobinamide on releasing NO; and (iv) combinations of cobinamide and cinaciguat. We will analyze the drugs' effects on bone formation and architecture, with micro-CT, histomorphometry, biomechanical testing, and gene expression profiling, and compare their effects on proliferation, differentiation, and survival of diabetic (pre)osteoblasts ex vivo. We will determine if mice expressing constitutively-active PKG1/2 and NOX4 knock-out mice are protected from diabetic bone loss, and examine if insulin mediates bone-protective effects via PKG, using (pre)osteoblast-specific PKG1/2 knock-out mice. These studies could be paradigm shifting, because they may define a novel mechanism for diabetic bone loss and provide a rational basis for testing cGMP-elevating drugs in diabetic osteoporosis, where they could provide a novel, anabolic treatment strategy.

描述（由适用提供）：糖尿病的骨质流失几乎没有引起人们的注意，特别是考虑到骨质疏松症影响约20％的1型糖尿病患者（T1DM）（T1DM）和骨质差的患者在2型糖尿病中也是一个问题。骨质流失是由于成骨细胞的形成减少，而不是骨质碎屑恢复的增加，因此，抗敏感性疗法不足。有缺陷的成骨细胞功能（S）的分子机制不是很好的定义，但可能包括过量产生活性氧（ROS）。一氧化氮（NO）在成骨细胞中起重要作用，临床试验表明，没有供体改善后绝经妇女的骨矿物质密度。但是，常规的没有供体会增强氧化应激。我们先前表明，NO/CGMP/蛋白激酶G（PKG）信号通路调节成骨细胞增殖和存活。我们发现，与对照小鼠相比，我们发现了增加的NADPH氧化酶（NOX4）表达，超过ROS的产生，降低了NO/CGMP的产生，并降低了具有链霉亲素诱导的T1DM的小鼠的骨骼和成骨细胞的PKG表达。用CGMP高度抗药性剂Cinaciguat处理，即使在高氧化应激下也具有活性，在很大程度上恢复了糖尿病（前）骨爆炸的有缺陷的增殖和分化，并改善了用T1DM的小鼠的骨形成和小梁骨体积。我们假设有缺陷的NO/CGMP/PKG信号 - 大专院部分，部分是途径酶 - 基因归因于糖尿病骨质流失的氧化损伤；恢复NO/CGMP信号传导和/或减少氧化应激可能是有效治疗糖尿病相关的骨质疏松症。具体目的是：（i）确定糖尿病（PRE）成骨细胞中NO/CGMP/PKG信号的机制和后果；（ii）分析CGMP升高药物对糖尿病诱导的体内骨质丧失的影响；（iii）检查pkg和nox4在糖尿病诱导的骨质流失中的作用。我们将研究糖尿病对NO合酶和鸟苷酸环化酶氧化和翻译后修饰以及成骨细胞中PKG转录调控的影响。我们将通过使用以下方式治疗小鼠来预防T1DM小鼠骨质流失的方法：（i）cinaciguat；（ii）核酰胺，维生素B12类似物和潜在的抗氧化剂；（iii）新颖的无供体硝基辛酰胺，它在释放NO时产生cobinamide；（iv）Cobinamide和cinaciguat的组合。我们将通过微观CT，组织倍率法，生物力学测试和基因表达分析分析药物对骨形成和结构的影响，并比较它们对糖尿病（PER）成骨成肌细胞的增殖，分化和生存的影响。我们将确定表达组成型PKG1/2和NOX4敲除小鼠的小鼠是否受到糖尿病性骨质流失的保护，并使用（PRE）成骨细胞特异性PKG1/2基因敲除小鼠检查胰岛素是否通过PKG介导骨保护作用。这些研究可能是范式转移，因为它们可能定义了一种新型的糖尿病骨质损失机制，并为在糖尿病性骨质疏松症中测试CGMP高度测试药物提供了合理的基础，它们可以提供一种新型的合成代谢治疗策略。