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：
9459312
负责人：
RENATE B PILZ
金额：
$ 37.66万
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 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9459312
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 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（由申请人提供）：令人惊讶的是，糖尿病引起的骨质流失很少受到关注，特别是考虑到约 20% 的 1 型糖尿病 (T1DM) 患者患有骨质疏松症，而且由于骨质疏松，骨质疏松也是 2 型糖尿病的一个问题。成骨细胞骨形成，而不是破骨细胞吸收增加，因此，抗吸收治疗是不够的。成骨细胞功能缺陷的分子机制尚未明确，但可能包括过度生成。活性氧 (ROS) 在成骨细胞中起着重要作用，临床试验表明，NO 供体可以改善绝经后妇女的骨矿物质密度；然而，我们之前的研究表明，NO 供体会增强氧化应激。我们发现 NO/cGMP/蛋白激酶 G (PKG) 信号通路调节成骨细胞增殖和存活，NADPH 氧化酶 (NOX4) 表达增加，ROS 生成过多，NO/cGMP 生成减少，并减少。与对照小鼠相比，链脲佐菌素诱导的 T1DM 小鼠的骨骼和成骨细胞中 PKG 的表达采用 cGMP 升高剂 cinaciguat 进行治疗，该药物即使在高氧化应激下也具有活性，可在很大程度上恢复糖尿病（前）骨的缺陷性增殖和分化。我们发现，NO/cGMP/PKG 信号传导缺陷在一定程度上是继发性的。途径酶的氧化损伤导致糖尿病中的骨质流失；恢复 NO/cGMP 信号传导和/或减少氧化应激可能是糖尿病相关骨质疏松症的有效治疗方法。糖尿病（前）成骨细胞中 NO/cGMP/PKG 信号受损的研究；（ii）分析 cGMP 升高剂对糖尿病引起的体内骨质流失的影响；研究 PKG 和 NOX4 在糖尿病引起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的影响，以及对成骨细胞中 PKG 转录调控的影响我们将比较几种预防骨质流失的方法。通过使用以下药物治疗患有 T1DM 的小鼠：(i) 西那西呱；(ii) 可宾酰胺，一种维生素 B12 类似物和强效抗氧化剂；新型 NO 供体亚硝酰基可宾酰胺，可在释放 NO 时生成可宾酰胺；以及 (iv) 可宾酰胺和 cinaciguat 的组合。我们将通过显微 CT、组织形态学、生物力学测试和基因分析药物对骨形成和结构的影响。表达谱，并比较它们对离体糖尿病（前）成骨细胞的增殖、分化和存活的影响。我们将确定小鼠表达是否具有组成型活性。 PKG1/2 和 NOX4 敲除小鼠免受糖尿病性骨质流失，并使用（前）成骨细胞特异性 PKG1/2 敲除小鼠检查胰岛素是否通过 PKG 介导骨骼保护作用。因为它们可能定义了糖尿病骨质流失的新机制，并为测试糖尿病骨质疏松症中的 cGMP 升高药物提供了合理的基础，从而提供了一种新的合成代谢治疗策略。