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

 描述(申请人提供)：糖尿病患者的骨丢失受到的关注少得令人惊讶，特别是考虑到骨质疏松症影响到大约20%的1型糖尿病(T1 DM)患者，而骨骼质量差也是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，抗吸收治疗是不够的。成骨细胞功能缺陷的分子机制(S)尚不清楚，但可能包括过度产生活性氧物种(ROS)。一氧化氮(NO)在成骨细胞中起着重要作用，临床试验表明，NO供体可提高绝经后妇女的骨密度；然而，传统的NO供体会增加氧化应激。我们先前发现，NO/cGMP/蛋白激酶G(PKG)信号通路调节成骨细胞的增殖和存活。我们发现，与对照组相比，链脲佐菌素诱导的T1 DM小鼠骨和成骨细胞中NADPH氧化酶(NOX4)表达增加，ROS生成过量，NO/cGMP生成减少，PKG表达减少。CGMP升高剂Cinaciguat即使在高氧化应激下也是活跃的，在很大程度上恢复了糖尿病(Pre)成骨细胞的缺陷增殖和分化，并改善了T1 DM小鼠的骨形成和骨小梁体积。我们假设，NO/cGMP/PKG信号通路的缺陷--部分继发于途径酶的氧化损伤--导致糖尿病骨丢失；恢复NO/cGMP信号通路和/或减少氧化应激可能是治疗糖尿病相关性骨质疏松症的有效方法(S)。其具体目的是：(I)确定糖尿病(Pre)成骨细胞中NO/cGMP/PKG信号受损的机制和后果；(Ii)分析cGMP增高剂在体内糖尿病诱导的骨丢失中的作用；(Iii)研究PKG和NOX4在糖尿病诱导的骨丢失中的作用。我们将研究糖尿病对成骨细胞一氧化氮合酶和鸟苷环化酶氧化和翻译后修饰的影响，以及对PKG转录调控的影响。我们将比较几种预防T1 DM小鼠骨质丢失的方法，方法是：(I)Cinaciguat；(Ii)椰子酰胺，一种维生素B12类似物，有效的抗氧化剂；(Iii)新型NO供体亚硝酰钴胺，它在释放NO时产生Cobin酰胺；以及(Iv)椰子酰胺和Cinaciguat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骨结构的影响，并比较它们对糖尿病(前)成骨细胞体外增殖、分化和存活的影响。我们将确定表达结构性活性PKG1/2和NOX4基因敲除小鼠是否受到糖尿病骨丢失的保护，并使用(前)成骨细胞特异性PKG1/2基因敲除小鼠来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的转变，因为它们可能定义一种新的糖尿病骨丢失机制，并为测试升高cGMP的药物治疗糖尿病骨质疏松症提供合理的基础，从而提供一种新的合成代谢治疗策略。

 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.