RESUMO
Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10â ng/ml) and interleukin-1ß (1â ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30â µmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.
RESUMO
Bone morphogenetic protein (BMP), an osteoinductive factor, is a cytokine that induces osteoblast differentiation and mineralization, and expected to be applicable for hard tissue reconstruction. Kielin/chordin-like protein (Kcp), a member of the family of cysteine-rich proteins, enhances BMP signaling. The present study found that expression of Kcp in osteoblasts was induced by BMP-2 in a concentration- and time-dependent manner. Up-regulation of Kcp by BMP-2 was inhibited by Dorsomorphin, a SMAD signaling inhibitor. The involvement of up-regulation of Kcp by BMP-2 in induction of osteoblast differentiation by BMP-2 was also examined, which showed that suppression of Kcp expression by si Kcp partially inhibited induction of osteoblast differentiation and mineralization by BMP-2. Together, these results suggest that Kcp induced by BMP-2 functions to provide positive feedback for promotion of osteoblastic differentiation and mineralization by BMP-2 in osteoblasts.