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

 描述（由申请人提供）：令人惊讶的是，糖尿病中的骨丢失很少受到关注，特别是考虑到骨质疏松症影响约20%的1型糖尿病（T1 DM）患者，并且骨质差也是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，而不是骨吸收增加，因此，抗骨吸收治疗是不够的。成骨细胞功能缺陷的分子机制尚未明确，但可能包括活性氧（ROS）的过度产生。一氧化氮（NO）在成骨细胞中起重要作用，临床试验表明NO供体改善绝经后妇女的骨密度;然而，常规NO供体增强氧化应激。我们以前表明，NO/cGMP/蛋白激酶G（PKG）信号通路调节成骨细胞的增殖和生存。我们发现，与对照小鼠相比，链脲佐菌素诱导的T1 DM小鼠的骨骼和成骨细胞中NADPH氧化酶（NOX 4）表达增加，ROS产生过量，NO/cGMP产生减少，PKG表达减少。cGMP升高剂西那西呱（即使在高氧化应激下也具有活性）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T1 DM小鼠的骨形成和骨小梁体积。我们假设，有缺陷的NO/cGMP/PKG信号通路-继发于氧化损伤的途径酶-有助于糖尿病的骨丢失;恢复NO/cGMP信号通路和/或减少氧化应激可能是有效的治疗糖尿病相关的骨质疏松症。具体目标是：（i）确定糖尿病（前）成骨细胞中受损的NO/cGMP/PKG信号传导的机制和后果;（ii）分析cGMP升高剂对体内糖尿病诱导的骨丢失的影响;和（iii）检查PKG和NOX 4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中NO合酶和鸟苷酸环化酶氧化和翻译后修饰以及PKG转录调控的影响。我们将比较几种方法，以防止骨质流失的小鼠与T1 DM治疗小鼠：（i）西那西呱;（ii）cobinamide，维生素B12类似物和有效的抗氧化剂;（iii）新的NO供体亚硝酰基-cobinamide，它产生cobinamide释放NO;和（iv）cobinamide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来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）成骨细胞增殖、分化和存活的影响。我们将使用（前）成骨细胞特异性PKG 1/2基因敲除小鼠，确定表达组成型活性PKG 1/2和NOX 4基因敲除小鼠是否受到糖尿病骨丢失的保护，并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变，因为它们可能定义了糖尿病骨丢失的新机制，并为测试糖尿病骨质疏松症中cGMP升高药物提供了合理的基础，在那里它们可以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.