Hyperglycemia increases the expression levels of sclerostin in a reactive oxygen species- and tumor necrosis factor-alpha-dependent manner

PURPOSE: Sclerostin, an inhibitor of Wnt/beta-catenin signaling, exerts negative effects on bone formation and contributes to periodontitis-induced alveolar bone loss. Recent studies have demonstrated that serum sclerostin levels are increased in diabetic patients and that sclerostin expression in alveolar bone is enhanced in a diabetic periodontitis model. However, the molecular mechanism of how sclerostin expression is enhanced in diabetic patients remains elusive. Therefore, in this study, the effect of hyperglycemia on the expression of sclerostin in osteoblast lineage cells was examined. METHODS: C2C12 and MLO-Y4 cells were used in this study. In order to examine the effect of hyperglycemia, the glucose concentration in the culture medium was adjusted to a range of levels between 40 and 100 mM. Gene expression levels were examined by quantitative reverse transcription-polymerase chain reaction and Western blot assays. Top-Flash reporter was used to examine the transcriptional activity of the beta-catenin/lymphoid enhanced factor/T-cell factor complex. Tumor necrosis factor-alpha (TNFalpha) protein levels were examined with the enzyme-linked immunosorbent assay. The effect of reactive oxygen species on sclerostin expression was examined by treating cells with 1 mM H2O2 or 20 mM N-acetylcysteine. RESULTS: The high glucose treatment increased the mRNA and protein levels of sclerostin. High glucose suppressed Wnt3a-induced Top-Flash reporter activity and the expression levels of osteoblast marker genes. High glucose increased reactive oxygen species production and TNFalpha expression levels. Treatment of cells with H2O2 also enhanced the expression levels of TNFalpha and sclerostin. In addition, N-acetylcysteine treatment or knockdown of TNFalpha attenuated high glucose-induced sclerostin expression. CONCLUSIONS: These results suggest that hyperglycemia increases sclerostin expression via the enhanced production of reactive oxygen species and TNFalpha.

Erratum: Institutions, Correspondence, Figures & Legends Correction. Hyperglycemia increases the expression levels of sclerostin in a reactive oxygen species- and tumor necrosis factor-alpha-dependent manner

Some parts of published paper were misprinted.

Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation

TNF-alpha, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-alpha signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-alpha-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-alpha down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-alpha induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-alpha. TNF-alpha activated the NF-kappaB and the JNK pathways. Inhibition of NF-kappaB or JNK activation reduced the inhibitory effect of TNF-alpha on ALP expression, whereas TNF-alpha-induced Msx2 expression was only suppressed by the inhibition of the NF-kappaB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-alpha on BMP2-regulated osteoblast differentiation and that the TNF-alpha-activated NF-kappaB pathway is responsible for Msx2 induction.

Trichostatin A-mediated upregulation of p21

Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21(WAF1) upregulation in TSA-treated osteoclasts, shRNA against p21(WAF1)-containing lentivirus was introduced into osteoclasts. The suppression of p21(WAF1) decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21(WAF1), and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.

High extracellular Ca2+ alone stimulates osteoclast formation but inhibits in the presence of other osteoclastogenic factors

High ambient Ca2+ at bone resorption sites have been implicated to play an important role in the regulation of bone remodeling. The present study was performed to clarify the mode of high extracellular Ca2+ (Ca2+e)-induced modulation of osteoclastogenesis and the expression of receptor activator of nuclear factor-kB ligand (RANKL) and osteoprotegerin (OPG), thereby to define its role in osteoclast formation. Mouse bone marrow cells were cocultured with osteoblastic cells in the absence or presence of osteoclastogenic factors such as 1,25-dihydroxyvitaminD3 (1,25-(OH)2vitD3) and macrophage colony-stimulating factor/soluble RANKL. Ca2+ concentration in media (1.8 mM) was adjusted to 3, 5, 7 or 10 mM. Osteoclast formation was confirmed by the appearance of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells and the expression of osteoclast phenotypic markers (calcitonin receptor, vitronectin receptor, cathepsin K, matrix metalloproteinase-9, carbonic anhydrase 2). High Ca2+e alone significantly stimulated osteoclast formation in a dose-dependent manner. However, in the presence of highly osteoclastogenic factors, high Ca2+e significantly inhibited osteoclastogenesis. High Ca2+e alone continuously up-regulated RANKL expression while only transiently increased OPG expression. However, in the presence of 1,25-(OH)2vitD3, high Ca2+e did not change the 1,25-(OH)2vitD3- induced RANKL expression while increased OPG expression. Taken together, these findings suggest that high Ca2+e alone increase osteoclastogenesis but inhibit in the presence of other osteoclastogenic factors. In addition, high Ca2+e-induced osteoclastogenesis may be mediated by osteoblasts via up-regulation of RANKL expression. Meanwhile up-regulated OPG might participate in the inhibitory effect of high Ca2+e on 1,25-(OH)2vitD3-induced osteoclastogenesis.