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...