Defective NO/cGMP/PKG Signaling Downregulates Sirt1 Expression in Aged Osteoblasts

Aging associated osteoporosis is a major health concern that develops following a progressive decline in bone volume and bone mineral density with increased fracture risk. Current osteoporosis therapies like parathyroid hormone and bisphosphonate treatment have significant side-effects like osteonecrosis of the jaw. Hence, better understanding of the pathophysiology of age-related bone loss is essential in identifying specific drug targets and developing novel therapeutic approaches to target osteoporosis in the elderly. Previous studies in our laboratory have shown that the nitric oxide/cyclic GMP (cGMP)/protein kinase G (PKG) pathway exerts a bone anabolic role in vitro in osteoblasts and osteocytes. Treatment with the cGMP elevating agent, Cinaciguat, protects against estrogen deficiency and type 1 diabetes associated osteoporosis. Our laboratory results have consistently shown reduced eNOS, PKG1 and PKG2 mRNA expression in the tibia of aged (12 month) compared to young (3 month) mice, indicating defective NO/cGMP/PKG signaling in the aged bone. Treatment of osteoblasts in vitro with 8-pCPT-cGMP (100 µM) induced Sirtuin 1 (Sirt 1) and its upstream transcriptional regulator, Nrf 2 mRNA expression. We suspect that defective NO/cGMP/PKG signaling in the aged bone results in the reduced Sirt1 and Nrf2 expression in the tibia of aged mice. Osteogenic differentiation studies (Alkaline phosphatase staining and gene expression analysis) from the isolated bone marrow stromal cells showed that the reduced osteogenic potential (ALP staining, and Runx2, Sirt1 gene expression) observed in the aged (wild type type) mice can be greatly improved by in vitro treatment with Cinaciguat and the NO-releasing agent, NO-Cobinamide. Transgenic mice with an osteoblast specific overexpression of PKG2 showed significantly increased osteogenic gene expression including Sirt1 and Nrf2 at 12 months of age. MicroCT analysis indicated that PKG2 overexpressing transgenic mice had a significant increase in bone volume, trabecular number and bone mineral density compared to the aged (12 month) wild type mice; providing protection from the age induced bone loss. The potential regulation of Sirt1/Nrf2 gene expression by the NO/cGMP/PKG2 signaling pathway is very exciting and lays the foundation for further studies directed at understanding the mechanism of this complex regulation. Our results provide a novel insight into the mechanism of bone loss in aging associated osteoporosis and indicate that cGMP elevating agents should be further studied as a prospective treatment