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

Abstract

PurposeSclerostin, an inhibitor of Wnt/β-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.MethodsC2C12 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 β-catenin/lymphoid enhanced factor/T-cell factor complex. Tumor necrosis factor-alpha (TNFα) 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.ResultsThe 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 TNFα expression levels. Treatment of cells with H2O2 also enhanced the expression levels of TNFα and sclerostin. In addition, N-acetylcysteine treatment or knockdown of TNFα attenuated high glucose-induced sclerostin expression.ConclusionsThese results suggest that hyperglycemia increases sclerostin expression via the enhanced production of reactive oxygen species and TNFα.Graphical Abstract

Sclerostin does not play a major role in the pathogenesis of skeletal complications in type 2 diabetes mellitus

“…It has been suggested that high glucose concentrations stimulate sclerostin secretion in osteoblastic and osteocytic cell lines. Consistent with previous data, our results show increased sclerostin production in UMR-106 cells with increasing glucose concentrations, implying a stimulatory effect of hyperglycaemia on sclerostin production [35, 36]. However, we could not detect sclerostin expression in the osteocytic cell line IDG-SW3 at any stage of differentiation, suggesting that IDG-SW3 cells might not be suitable for the study of sclerostin expression.…”

VEGF Expression in Diabetic Rats Promotes Alveolar Bone Resorption by <i>Porphyromonas gingivalis</i> LPS

“…In cytokine imbalance caused by hyperglycemia, it has been extensively reported that inflammatory cytokines, such as TNF- 10) , IL-1 11) , and IL-6 11) , increase. These inflammatory cytokines both directly and indirectly promote differentiation and activation of osteoclasts 13) .…”

“…In a previous study, we have reported that expression of the angiogeneic factor VEGF in the periodontium is increased in rats with diabetes mellitus when compared to that in healthy rats 22) . Hyperglycemia-induced increases in levels of inflammatory cytokines, such as TNF- 10) , IL-1 11) and IL-6 11) , cause progression of periodontitis in diabetes mellitus. However, how VEGF, a cytokine expressed and present in the periodontium under hyperglycemic conditions, affects bone resorption caused by periodontitis is unknown.…”

“…Hyperglycemia causes formation and accumulation of irreversible advanced glycation end products (AGEs) in the body 31) and induces expression of their primary receptor RAGE 32) . Interactions between the AGEs and RAGE result in the dysfunction of immune cells 6,7) and functional alterations of various cells 8,9) , causing cytokine imbalance associated with inflammatory cytokine increases 10,11) . Furthermore, hyperglycemia shifts the balance of the RANKL/OPG interaction in the direction of tissue destruction through AGEs/RAGE interaction 33,34) .…”

“…Among them, the main mechanisms involve immune dysfunction 6,7 ) , cellular stress 8,9 ) , and cytokine imbalance 10,11) caused by hyperglycemia, which enhances tissue destruction of periodontitis and impairs tissue repair. The mechanism of exacerbation of alveolar bone resorption due to cytokine imbalance is initiated when hyperglycemia increases the level of inflammatory cytokines, such as TNF- 10) , IL-1 11) , and IL-6 11) . These cytokines affect the interaction between RANKL and OPG 12) , and as a result, osteoclast differentiation is promoted.…”