J Periodontal Implant Sci.  2015 Jun;45(3):101-110. 10.5051/jpis.2015.45.3.101.

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

Affiliations
  • 1Department of Molecular Genetics, School of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea. gskim@snu.ac.kr, baekjh@snu.ac.kr
  • 2Department of Anatomy, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
  • 3Department of Pharmacology, College of Dentistry, Gangneung-Wonju National University, Gangneung, Korea.

Abstract

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.

Keyword

Hyperglycemia; Osteoblasts; Reactive oxygen species; Tumor necrosis factor-alpha

MeSH Terms

Acetylcysteine
Alveolar Bone Loss
Blotting, Western
Enzyme-Linked Immunosorbent Assay
Gene Expression
Glucose
Humans
Hyperglycemia*
Necrosis*
Osteoblasts
Osteogenesis
Oxygen*
Periodontitis
Reactive Oxygen Species
RNA, Messenger
Tumor Necrosis Factor-alpha
Acetylcysteine
Glucose
Oxygen
RNA, Messenger
Reactive Oxygen Species
Tumor Necrosis Factor-alpha

Figure

  • Figure 1 High glucose increases sclerostin expression. (A-C) C2C12 cells were cultured in osteogenic medium in the presence or absence of high glucose for 48 hours (h) unless specified, and (A) RT-PCR and (B) Western blot analysis were performed. (D, E) MLO-Y4 cells were incubated for 48 hours in the presence or absence of high glucose treatment, followed by (D) quantitative RT-PCR and (E) Western blot analysis. The graphs (A, D) indicate the mean±standard deviation of the triplicate samples (c)P<0.001, d)P<0.0001). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction. In the Figure 1C, 5 and 100 mM indicate the concentration of glucose in the culture medium.

  • Figure 2 High glucose negatively regulates Wnt/β-catenin signaling. (A) C2C12 cells were incubated for 24 hours in the presence or absence of Wnt3a (50 ng/mL) or high glucose (100 mM), followed by quantitative reverse transcription-polymerase chain reaction of osteogenic marker genes. (B) MLO-Y4 cells were transiently transfected with the β-catenin expression vector and Top-Flash luciferase reporter and cultured in the presence or absence of Wnt3a or high glucose (40 mM) for 72 hours. The graphs indicate the mean±standard deviation of (A) the triplicate or (B) quadruplicate samples (a)P<0.05, c)P<0.001, d)P<0.0001). In the figures, 5 and 100 mM indicate the concentration of glucose in the culture medium.

  • Figure 3 High glucose enhances reactive oxygen species production. Dichlorofluorescein diacetate was added to the culture medium, and (A) C2C12 and (B) MLO-Y4 cells were treated with high glucose (100 mM) or H2O2 (1 mM) for the indicated time periods, followed by measuring fluorescence from oxidized dichlorofluorescein. The graphs indicate the mean±standard deviation of the octuplicate samples (c)P<0.001, d)P<0.0001; significantly different from the control group at each time point). DCF, dichlorofluorescein; CON, control; HG, high glucose; m, minutes; h, hours.

  • Figure 4 Reactive oxygen species contribute to high glucose-induced sclerostin expression. (A) C2C12 cells were incubated for 48 hours in the presence of H2O2 at the indicated concentrations, followed by (B-E) quantitative RT-PCR for sclerostin. (B, D) C2C12 and (C, E) MLO-Y4 (C, E) cells were incubated for 48 hours in the presence or absence of the indicated reagents, followed by (B, C) quantitative RT-PCR and (D, E) Western blot analysis (D, E). The graphs (A-C) indicate the mean±standard deviation of the three to five samples (b)P<0.01, d)P<0.0001). CON (control), 5 mM glucose; HG (high glucose), 100 mM glucose; H2O2, 1 mM H2O2; NAC, 20 mM N-acetylcysteine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction.

  • Figure 5 High glucose and reactive oxygen species increase TNFα expression. (A, C) C2C12 and (B, D) MLO-Y4 cells were incubated for 48 hours in the presence of the indicated reagents. (A, B) An ELISA assay for TNFα was then performed using cell lysates, and (C, D) RT-PCR for TNFα was performed. The graphs indicate the mean±standard deviation of the three to eight samples (a)P<0.05, b)P<0.01, c)P<0.001, d)P<0.0001). TNFα, tumor necrosis factor-alpha; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase chain reaction; CON, control; HG, high glucose; VEH, vehicle; NAC; N-acetylcysteine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

  • Figure 6 TNFα mediates high glucose-induced sclerostin expression. (A, B, E) C2C12 and (C, D, F) MLO-Y4 cells were transiently transfected with control siRNA or TNFα siRNA, followed by incubation for 48 hours in the presence or absence of the indicated reagents. (A-D) Quantitative RT-PCR for TNFα and sclerostin, and (E, F) Western blot analysis were then performed. The graphs indicate the mean±standard deviation of the triplicate samples (a)P<0.05, b)P<0.01, c)P<0.001, d)P<0.0001). TNFα, tumor necrosis factor-alpha; RT-PCR, reverse transcription-polymerase chain reaction; CON, control; HG, high glucose; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


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Jakob Starup-Linde, Katrine Hygum, Bente Lomholt Langdahl
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