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

总结 骨质疏松症影响着数百万的美国男性和女性，是导致骨折的主要原因。 50岁以上的人。骨丢失主要是由于骨形成受损，但FDA批准的骨- 合成代谢作用（PTH类似物和硬化素中和抗体romosozumab）具有严重的局限性。 我们已经发现NO/cGMP/蛋白激酶G（PKG）信号传导的药理学或遗传刺激 途径增强糖尿病和老年小鼠的骨形成并防止骨丢失;其机制是通过 增加Wnt/β-catenin信号传导和减少氧化应激。沉默信息调节子T1（Sirtuin 1，SIRT 1） 其底物NAD+随着年龄的增长而减少，导致许多与年龄有关的疾病，包括骨质疏松症。 成骨细胞特异性Sirt 1基因敲除小鼠骨质疏松，而Sirt 1过表达小鼠具有高骨量。 我们最近发现PKG激活增加了成骨细胞、间充质干细胞和成纤维细胞中SIRT 1的表达， （MSC）和老年小鼠的骨通过稳定Sirt 1 mRNA; RNA结合蛋白hnRNPA 1是必需的。 已知SIRT 1增加NO合成，可能导致PKG的正反馈回路。支持 PKG和SIRT 1之间的正反馈，我们发现它们协同增加促生存基因， 成骨细胞和防止氧化应激诱导的细胞凋亡。作为我们对NO/cGMP/PKG信号传导的兴趣的一部分， 我们已经开发了亚硝酰基-可宾酰胺（NO-Cbi），一种也是强抗氧化剂的NO供体。最近的工作 显示10周的NO-Cbi疗程改善了24个月大的骨形成、结构和强度， 小鼠拟议研究的总体目标是确定骨骼中PKG和SIRT 1之间的相互作用， 并基于PKG和SIRT 1激活剂设计了一种治疗年龄相关性骨质疏松症的新策略。在Aim中 1我们将定义PKG如何增加MSC和成骨细胞中的Sirt 1 mRNA，重点关注PKG对 hnRNPA 1。在目标2中，我们将确定PKG-SIRT 1串扰在老年小鼠骨骼中的后果。我们 将确定PKG是否需要SIRT 1来改善骨形成和强度，通过检查 PKG激活剂对条件性成骨细胞特异性Sirt 1基因敲除小鼠骨的影响为了确定SIRT 1- 诱导的NO合成和PKG激活是SIRT 1激活剂在骨中的积极作用所必需的，我们 将检查这些药物对成骨细胞中Prkg 2条件性缺失小鼠骨骼的影响。在Aim中 3，我们将测试组合PKG-和SIRT 1-活化剂是否可以协同增加骨形成 在老年小鼠中，与单独的每种治疗相比。我们将使用NO-Cbi和鸟苷酸环化酶激活剂 cinaciguat激活PKG，并直接SIRT 1激活剂和烟酰胺核苷（NAD+前体）激活 SIRT 1.拟议的工作将填补重要的知识空白，定义PKG-SIRT 1串扰在骨中的作用， 在衰老过程中，并确定PKG-SIRT 1如何减少骨骼中与年龄相关的氧化应激;重要的是， 这项工作为开发新的骨合成代谢疗法治疗年龄相关性骨质疏松症奠定了基础。

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.