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

• 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
• 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

 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，特别是考虑到骨质疏松症影响约 20% 的 1 型糖尿病 (T1DM) 患者，而骨质量差也是 2 型糖尿病的一个问题。骨丢失是由于成骨细胞骨形成减少，而不是破骨细胞吸收增加，因此，抗吸收疗法是不够的。成骨细胞功能缺陷的分子机制尚未明确，但可能包括活性氧（ROS）的过度产生。一氧化氮 (NO) 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，传统的一氧化氮供体会增强氧化应激。我们之前表明 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，链脲佐菌素诱导的 T1DM 小鼠的骨骼和成骨细胞中 NADPH 氧化酶 (NOX4) 表达增加、ROS 生成过多、NO/cGMP 生成减少以及 PKG 表达减少。即使在高氧化应激下也具有活性的 cGMP 升高剂 cinaciguat 治疗可在很大程度上恢复糖尿病（前）成骨细胞的增殖和分化缺陷，并改善 T1DM 小鼠的骨形成和骨小梁体积。我们假设 NO/cGMP/PKG 信号传导缺陷（部分继发于途径酶的氧化损伤）导致糖尿病患者骨质流失；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。具体目标是：(i) 确定糖尿病（前）成骨细胞中 NO/cGMP/PKG 信号传导受损的机制和后果； (ii) 分析 cGMP 升高剂对糖尿病引起的体内骨质流失的影响； (iii) 检查 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 T1DM 小鼠骨质流失的方法，方法是用以下药物治疗小鼠：(i) cinaciguat； (ii) Cobinamide，一种维生素 B12 类似物和有效的抗氧化剂； (iii)新型NO供体亚硝酰基钴酰胺，其在释放NO时生成钴酰胺； (iv)可滨酰胺和西那西呱的组合。我们将通过显微 CT、组织形态计量学、生物力学测试和基因表达谱分析药物对骨形成和结构的影响，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。我们将确定表达组成型活性 PKG1/2 的小鼠和 NOX4 敲除小鼠是否可以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。这些研究可能会带来范式转变，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，从而提供了一种新颖的合成代谢治疗策略。