Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. Compared with the well established role of TNF in osteoclastogenesis, mechanisms by which TNF inhibits osteoblast function have not been fully determined. Runx2 is an osteoblast-specific transcription factor whose steady-state protein levels are regulated by proteasomal degradation, mediated by the E3 ubiquitin ligases, Smurf1 and Smurf2. We hypothesized that TNF inhibits osteoblast function through Smurf-mediated Runx2 degradation. We treated C2C12 and 2T3 osteoblast precursor cell lines and primary osteoblasts with TNF and found that TNF, but not interleukin-1, significantly increased Smurf1 and Smurf2 expression. TNF increased the degradation of endogenous or transfected Runx2 protein, which was blocked by treating cells with a proteasomal inhibitor or by infecting cells with small interfering (si)RNA against Smurf1 or Smurf2. TNF inhibited the expression of bone morphogenetic protein and transforming growth factor-␤ signaling reporter constructs, and the inhibition of each was blocked by Smurf1 siRNA and Smurf2 siRNA, respectively. Overexpression of Smurf1 and/or Smurf2 siRNAs prevented the inhibitory effect of TNF on Runx2 reporter. Consistent with these in vitro findings, bones from TNF transgenic mice or TNF-injected wild type mice had increased Smurf1 and decreased Runx2 protein levels. We propose that one of the mechanisms by which TNF inhibits bone formation in inflammatory bone disorders is by promoting Runx2 proteasomal degradation through up-regulation of Smurf1 and Smurf2 expression. Tumor necrosis factor (TNF)2 is a major contributor to pathologic bone loss through stimulation of osteoclastic bone resorption and inhibition of osteoblastic bone formation. In patients with rheumatoid arthritis, TNF and other cytokines are overproduced in inflamed joints by various cells infiltrating the synovial membrane. This leads to severe local erosion of cartilage and bone, periarticular osteopenia, as well as systemic osteoporosis (1, 2). Under these conditions, osteoblasts do not catch up with the accelerated bone resorption, indicating impaired osteoblast function (3). The inhibitory effects of TNF on bone formation in vitro were first described in 1987 in neonatal rat calvarial organ cultures (4). Subsequent studies demonstrated that TNF inhibits recruitment of osteoblast progenitors, reduces expression of genes produced by mature osteoblasts, and promotes osteoblast apoptosis through nuclear factor-B signaling pathway (5-9). However, compared with our understanding of the role of TNF in osteoclast biology, little is known of the molecular mechanisms that mediate the effect of TNF on osteoblast inhibition.To date, the most important mechanistic finding of TNF-mediated osteoblast inhibition was the demonstration that TNF decreases Runtrelated gene 2 (Runx2) expression and its DNA binding activity in osteoblasts (10...

Runx2 is a bone-specific transcription factor that plays a critical role in bone development, postnatal bone formation, and chondrocyte maturation. The protein levels of Runx2 are regulated by the ubiquitin-proteasome pathway. In previous studies we discovered that E3 ubiquitin ligase Smad ubiquitin regulatory factor 1 (Smurf1) induces Runx2 degradation in a ubiquitin-proteasome-dependent manner, and Smurf1 plays an important role in osteoblast function and bone formation. In the present studies we investigated the molecular mechanism of Smurf1-induced Runx2 degradation. Smurf1 interacts with the PY motif of substrate proteins, and a PY motif has been identified in the C terminus of the Runx2 protein. To determine whether Smurf1 induces Runx2 degradation through the interaction with the PY motif of Runx2, we created a mutant Runx2 with a PY motif deletion and found that Smurf1 retained some of its ability to induce the degradation of the mutant Runx2, suggesting that Smurf1 could induce Runx2 degradation through an indirect mechanism. Smurf1 has been shown to interact with Smads 1, 5, 6, and 7, and Smads 1 and 5 also interact with Runx2. In the present studies we found that Smads 1 and 5 had no effect on Smurf1-induced Runx2 degradation. Although Smads 6 and 7 bind Smurf1, it is not known if Smads 6 or 7 interacts with Runx2 and mediate Runx2 degradation. We performed immunoprecipitation assays and found that Smad6 but not Smad7 interacts with Runx2. Smad6 enhances Smurf1-induced Runx2 degradation in an ubiquitin-proteasome-dependent manner. These results demonstrate that in addition to its interaction with the PY motif of Runx2, Smurf1 induces Runx2 degradation in a Smad6-dependent manner. Smurf1-induced Runx2 degradation serves as a negative regulatory mechanism for the BMP-Smad-Runx2 signaling pathway.Runx2 (Runt-related gene 2) is a bone-specific transcription factor that belongs to the runt-domain gene family. DNA-binding sites for Runx2 have been identified in the promoter regions of many osteoblastspecific genes (1-6), and Runx2 binds responsive elements in these promoters and regulates the transcription of these genes. Targeted disruption of Runx2 in mice reveals that Runx2 expression is absolutely required for bone development in vivo. Homozygous Runx2-deficient mice die soon after birth due to an inability to breathe. The most pronounced effect is a complete lack of both endochondral and intramembranous ossification (7,8), with an absence of mature osteoblasts throughout the body. Heterozygous mutant mice have skeletal abnormalities similar to those seen in a human mutation called cleidocranial dysplasia syndrome (9, 10) and delayed development of intramembranous bones (7,8). In transgenic mice overexpressing a dominant-negative Runx2 DNA binding domain (mRunx2) driven by the osteocalcin promoter, skeletons are normal at birth, but the mice suffer from osteopenia due to a decrease in bone formation rate 3 weeks after birth (11). These results indicate that Runx2 plays a crucial role not only in bone de...

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.

C-type natriuretic peptide (CNP) stimulates the differentiation and inhibits the proliferation of osteoblastic lineage cells. In this study, we examined whether the effects of CNP on osteoblastic functions change with aging using calvarial osteoblast-like cells from 25-week-old (young) and 120-week-old (aged) rats. CNP inhibited DNA synthesis and stimulated collagen synthesis and mineralized bone nodule formation. These effects were less pronounced in aged rat cells, suggesting the age-related attenuation of CNP-induced signaling. They were also blocked by the treatment of young rat cells with KT5823, a protein kinase G (PKG) inhibitor, but not by the treatment of aged rat cells with KT5823. CNP stimulated cGMP production in young rat cells, but not in aged rat cells. Natriuretic peptide receptor (NPR)-B, which has a guanylyl cyclase activity domain, and NPR-C, which has no enzyme activity domain, were predominantly expressed in young and aged rat cells, respectively. C-ANF, an NPR-C agonist, mimicked the effects of CNP on the proliferation and differentiation of aged rat cells; these effects were inhibited by the treatment with pertussis toxin (PTX), a Gi protein inhibitor. CNP and C-ANF evoked intracellular levels of inositol-1,4,5-triphosphate and Ca(2+), which are markers for phospholiase C (PLC) activation, in aged rat cells, and the effects of these two peptides were also blocked by the treatment with PTX. From these results, we concluded that CNP acts as a positive regulator of bone formation by osteoblasts and that the signaling pathway for CNP is switched from NPR-B/cGMP/PKG to NPR-C/G(i) protein/PLC with aging.

The effects of prostaglandin E2 (PGE2) on the proliferation and differentiation of osteoblastic cells were studied in osteoblast-like cells isolated from adult rat calvaria. Treatment of the cells with PGE2 within the concentration range 10(-8)-10(-5) M resulted in a dose-dependent increase in alkaline phosphatase (ALP) activity, [3H]proline incorporation into collagenase-digestible protein, and mineralized bone nodule (BN) formation, as well as a dose-dependent decrease in [3H]thymidine incorporation into the cells. PGE2 also caused a dose-dependent increase in the intracellular cyclic adenosine monophosphate (cAMP) content, with a maximal effective concentration of 10(-5) M; this effect of PGE2 was mimicked by forskolin, an adenylate cyclase activator. The treatment of adult calvarial cells with forskolin decreased BN formation, ALP activity, and collagen synthesis. These results suggested that cAMP does not have a stimulatory, but rather a suppressive, effect on the differentiation of adult rat calvarial cells. A time-course study of cAMP accumulation showed that both PGE2- and forskolin-induced cAMP reached a maximum at 5 min after the treatment, but the former rapidly returned to the basal level by 40 min, while the latter declined slowly and was still at 70% of the maximal level at 60 min, suggesting that PGE2 activates phosphodiesterase as well as adenylate cyclase. The presence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, reduced the rate of degradation of cAMP formed after PGE2 treatment, suggesting the involvement of calmodulin in the activation of phosphodiesterase. However, PGE2 also caused the production of inositol 1,4,5-triphosphate (IP3) and an elevation of the intracellular Ca2+ concentration ([Ca2+]i), both of which peaked at 15 s and returned to the basal level within 1 min. Submaximal responses of the IP3 production and the [Ca2+]i elevation to PGE2 were obtained at 10(-5) M. W-7 decreased both basal and PGE2-induced ALP activity, collagen synthesis and BN formation, indicating the involvement of Ca2+/calmodulin-dependent protein kinase in the PGE2-induced differentiation of calvarial cells. From these results, we concluded that PGE2 inhibits the proliferation and stimulates the differentiation of calvarial osteoblasts by elevating the [Ca2+]i through the activation of a phosphoinositide turnover, but not via an activation of adenylate cyclase. We also found that BN formation varies, depending on the time of PGE2 addition, suggesting that responsiveness of the cells to PGE2 may change during the culture period.