PKG Regulation of Sirtuin 1 as a Novel Treatment Strategy for Age-related Osteoporosis

摘要 与年龄相关的骨质疏松症影响着数百万美国男性和女性，是美国人骨折的主要原因 那些50岁以上的人。骨丢失主要是由于骨形成障碍，但FDA批准的骨形成药物- 合成代谢作用(甲状旁腺素类似物和硬化素中和抗体romosozumab)有严重的局限性。 我们已经发现，药物或遗传刺激对NO/cGMP/蛋白激酶G(PKG)信号的影响 途径促进糖尿病和衰老小鼠的骨形成和防止骨丢失；其机制是通过 增加Wnt/β-连环蛋白信号转导，减少氧化应激。静音信息调节器T1(Sirtuin1、SIRT1) 其底物NAD+随着年龄的增长而减少，导致许多与年龄相关的疾病，包括骨质疏松症。 成骨细胞特异性Sirt1基因敲除的小鼠骨质疏松，而Sirt1过度表达的小鼠骨量较高。 我们最近发现，PKG的激活增加了成骨细胞、间充质干细胞中SIRT1的表达 (MSCs)，和衰老小鼠的骨骼通过稳定Sirt1 mRNA；RNA结合蛋白hnRNPA1是必需的。 已知SIRT1可增加NO的合成，可能导致与PKG的正反馈循环。支座 PKG和SIRT1之间的正反馈，我们发现它们协同增加了促生存基因 并防止氧化应激诱导的细胞凋亡。作为我们对NO/cGMP/PKG信号的兴趣的一部分， 我们已经开发出亚硝酰钴酰胺(NO-CBI)，这是一种NO供体，也是一种强大的抗氧化剂。近期工作 结果显示，在24个月大的婴儿中，10周的非CBI疗程改善了骨的形成、结构和强度 老鼠。拟议研究的总体目标是确定PKG和SIRT1在骨骼中的相互作用， 设计了一种基于PKG和SIRT1激活剂的治疗老年性骨质疏松症的新策略。在AIM 1我们将定义PKG如何增加MSCs和成骨细胞中Sirt1的mRNA，重点是PKG对 HnRNPA1.在目标2中，我们将确定PKG-SIRT1串扰在老年小鼠骨骼中的后果。我们 将通过研究PKG是否需要SIRT1来改善骨形成和强度 条件性成骨细胞特异性Sirt1基因敲除小鼠骨骼上的PKG激活剂。以确定SIRT1是否- 诱导NO合成和PKG活化是SIRT1激活剂在骨中发挥积极作用所必需的 将在成骨细胞中有条件地缺失Prkg2的小鼠中检查这些药物对骨骼的影响。在AIM 3，我们将测试联合使用PKG和SIRT1激活剂是否可以协同促进骨形成 在老年小鼠中，与每种单独处理相比。我们将使用NO-CBI和鸟苷酸环化酶激活剂 Cinaciguat激活PKG，并直接激活SIRT1激活剂和NAD+前体烟酰胺核苷 SIRT1。拟议的工作将填补重要的知识空白，确定PKG-SIRT1串扰在骨骼中的作用 在衰老过程中以及确定PKG-SIRT1如何减少骨骼中与年龄相关的氧化应激；重要的是， 所提出的工作为开发治疗老年性骨质疏松症的新型骨合成代谢疗法奠定了基础。

SUMMARY Age-related osteoporosis affects millions of American men and women, and is a major cause of fractures in those over age 50. Bone loss is due mainly to impaired bone formation, but FDA-approved agents with bone- anabolic effects (PTH analogs and the sclerostin-neutralizing antibody romosozumab) have serious limitations. We have found that pharmacologic or genetic stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway enhances bone formation and prevents bone loss in diabetic and aged mice; the mechanism is through increased Wnt/β-catenin signaling and reduced oxidative stress. Silent information regulator T1 (Sirtuin1, SIRT1) and its substrate NAD+ decrease with age, contributing to many age-related diseases, including osteoporosis. Osteoblast-specific Sirt1 knockout mice are osteoporotic while Sirt1 overexpressing mice have high bone mass. We found recently that PKG activation increases SIRT1 expression in osteoblasts, mesenchymal stem cells (MSCs), and bones of aged mice by stabilizing Sirt1 mRNA; the RNA binding protein hnRNPA1 is required. SIRT1 is known to increase NO synthesis, possibly leading to a positive feedback loop with PKG. Supporting positive feedback between PKG and SIRT1, we found they synergistically increase pro-survival genes in osteoblasts and prevent oxidative stress-induced apoptosis. As part of our interest in NO/cGMP/PKG signaling, we have developed nitrosyl-cobinamide (NO-Cbi), a NO donor that is also a strong anti-oxidant. Recent work shows that a 10 week course of NO-Cbi improved bone formation, architecture, and strength in 24 month-old mice. The overall goal of the proposed studies is to define the interactions between PKG and SIRT1 in bone, and devise a novel treatment strategy for age-related osteoporosis based on PKG and SIRT1 activators. In Aim 1 we will define how PKG increases Sirt1 mRNA in MSCs and osteoblasts, focusing on PKG regulation of hnRNPA1. In Aim 2, we will determine the consequences of PKG-SIRT1 crosstalk in bones of aged mice. We will determine whether PKG requires SIRT1 to improve bone formation and strength by examining the effects of PKG-activating agents on bone in conditional osteoblast-specific Sirt1 knockout mice. To determine if SIRT1- induced NO synthesis and PKG activation is necessary for the positive effects of SIRT1 activators in bone, we will examine the skeletal effects of these agents in mice with conditional deletion of Prkg2 in osteoblasts. In Aim 3, we will test whether combining PKG- and SIRT1-activating agents can synergistically increase bone formation in aged mice, compared to each treatment alone. We will use NO-Cbi and the guanylyl cyclase activator cinaciguat to activate PKG, and direct SIRT1 activators and nicotinamide riboside, a NAD+ precursor, to activate SIRT1. The proposed work will fill important knowledge gaps, defining the role of PKG-SIRT1 crosstalk in bone during aging and determining how PKG-SIRT1 reduce age-related oxidative stress in bone; importantly, the proposed work lays the foundation for developing novel bone-anabolic therapies for age-related osteoporosis.