IADR Abstract Archives
8-Nitro-cGMP, a downstream signaling molecule of nitric oxide, is a novel regulator of bone metabolism that suppresses mineralization by osteoblasts and enhances differentiation of osteoclasts
Objectives: It is known that nitric oxide (NO) activates soluble guanylate cyclase, and the resultant cGMP functions as a second messenger of NO. Recently, 8-nitro-cGMP was identified as a novel signaling molecule of NO. We reported that 8-nitro-cGMP formed in the growth plate cartilage promotes bone growth via promotion of chondrocyte proliferation. NO is also known to regulate bone remodeling composed of bone formation by osteoblasts and bone resorption by osteoclasts. Since we found that inflammatory cytokines enhanced 8-nitro-cGMP production in both osteoblasts and osteoclasts, we investigated the role of 8-nitro-cGMP in differentiation of osteoblasts and osteoclasts.
Methods: Mouse calvarial osteoblasts were cultured in the presence and absence of 8-nitro-cGMP or 8-bromo-cGMP, a membrane-permeable cGMP derivative. Alkaline phosphatase (ALP) activity of the cells and mineralization of the cultures were quantitatively assessed. RANKL-induced differentiation of mouse bone marrow macrophages into osteoclasts in the presence and absence of 8-nitro-cGMP or 8-bromo-cGMP was evaluated by activity staining for tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits. Expression of the differentiation marker genes of osteoblasts and osteoclasts was quantitatively assessed by real-time RT-PCR. The expression of CARS2, the enzyme that plays a central role in degradation of 8-nitro-cGMP, was suppressed by its siRNA.
Results: 8-Nitro-cGMP reduced ALP activity of and mineralization by osteoblasts, which was a marked contrast to that 8-bromo-cGMP enhanced them. Expression of the osteoblast marker genes was suppressed by 8-nitro-cGMP. In addition, Cars2 siRNA had the effects similar to that of 8-nitro-cGMP on osteoblast differentiation. On the other hand, 8-nitro-cGMP significantly promoted osteoclastogenesis induced by RANKL with enhanced expression of RANK, the receptor for RANKL. 8-Bromo-cGMP did not have an effect on osteoclastogenesis.
Conclusions: We found that 8-nitro-cGMP negatively and positively regulates differentiation of osteoblasts and osteoclasts, respectively, which might contribute to inflammatory bone loss including that in periodontitis.
Methods: Mouse calvarial osteoblasts were cultured in the presence and absence of 8-nitro-cGMP or 8-bromo-cGMP, a membrane-permeable cGMP derivative. Alkaline phosphatase (ALP) activity of the cells and mineralization of the cultures were quantitatively assessed. RANKL-induced differentiation of mouse bone marrow macrophages into osteoclasts in the presence and absence of 8-nitro-cGMP or 8-bromo-cGMP was evaluated by activity staining for tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits. Expression of the differentiation marker genes of osteoblasts and osteoclasts was quantitatively assessed by real-time RT-PCR. The expression of CARS2, the enzyme that plays a central role in degradation of 8-nitro-cGMP, was suppressed by its siRNA.
Results: 8-Nitro-cGMP reduced ALP activity of and mineralization by osteoblasts, which was a marked contrast to that 8-bromo-cGMP enhanced them. Expression of the osteoblast marker genes was suppressed by 8-nitro-cGMP. In addition, Cars2 siRNA had the effects similar to that of 8-nitro-cGMP on osteoblast differentiation. On the other hand, 8-nitro-cGMP significantly promoted osteoclastogenesis induced by RANKL with enhanced expression of RANK, the receptor for RANKL. 8-Bromo-cGMP did not have an effect on osteoclastogenesis.
Conclusions: We found that 8-nitro-cGMP negatively and positively regulates differentiation of osteoblasts and osteoclasts, respectively, which might contribute to inflammatory bone loss including that in periodontitis.