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DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter.

Papathanasiou I, Kostopoulou F, Malizos KN, Tsezou A - Arthritis Res. Ther. (2015)

Bottom Line: Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known.We observed that SOST's expression was upregulated in OA chondrocytes compared to normal.Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. ioanna_papathanasiou@yahoo.gr.

ABSTRACT

Introduction: Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST's expression in OA chondrocytes.

Methods: SOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5'-Aza-2-deoxycytidine (5-AzadC) on SOST's expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).

Results: We observed that SOST's expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.

Conclusions: We report novel findings that DNA methylation regulates SOST's expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes' expression observed in OA.

No MeSH data available.


Related in: MedlinePlus

Effect of 5-AzadC treatment on SOST expression and DNA methylation status in the CpG-rich region of the SOST promoter. a Quantitative SOST mRNA expression in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.041). b Representative western blot of SOST protein levels in cultured normal chondrocytes after treatment with 5 μM 5-AzadC and a bar graph showing relative SOST protein expression normalized to β-actin in 5-AzadC-treated normal chondrocytes (n = 3) (error bars = standard error, *p = 0.009). c DNA methylation and unmethylation status of the SOST promoter in cultured normal chondrocytes after treatment with 5 μM 5-AzadC. d DNA methylation values in CpG-rich region of the SOST promoter in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC by quantitative methylation-specific PCR (error bars = standard error, *p = 0.032)
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Fig3: Effect of 5-AzadC treatment on SOST expression and DNA methylation status in the CpG-rich region of the SOST promoter. a Quantitative SOST mRNA expression in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.041). b Representative western blot of SOST protein levels in cultured normal chondrocytes after treatment with 5 μM 5-AzadC and a bar graph showing relative SOST protein expression normalized to β-actin in 5-AzadC-treated normal chondrocytes (n = 3) (error bars = standard error, *p = 0.009). c DNA methylation and unmethylation status of the SOST promoter in cultured normal chondrocytes after treatment with 5 μM 5-AzadC. d DNA methylation values in CpG-rich region of the SOST promoter in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC by quantitative methylation-specific PCR (error bars = standard error, *p = 0.032)

Mentions: To confirm the causal inverse association between SOST promoter methylation and gene expression, we evaluated the ability of the demethylating agent 5-AzadC to promote SOST expression in chondrocytes. Normal chondrocytes, for which the promoter was found to be methylated, were treated with 5-AzadC and subsequently we evaluated SOST mRNA and protein levels by real-time PCR and western blot analysis, respectively. We found that SOST mRNA and protein levels (p = 0.041, p = 0.009, respectively) were markedly increased in 5-AzadC-treated cells compared to untreated (Fig. 3a, b) and this 5-AzadC-induced change in gene expression was associated with a decrease in DNA methylation in the CpG-rich region of the SOST promoter (p = 0.032) (Fig. 3c, d).Fig. 3


DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter.

Papathanasiou I, Kostopoulou F, Malizos KN, Tsezou A - Arthritis Res. Ther. (2015)

Effect of 5-AzadC treatment on SOST expression and DNA methylation status in the CpG-rich region of the SOST promoter. a Quantitative SOST mRNA expression in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.041). b Representative western blot of SOST protein levels in cultured normal chondrocytes after treatment with 5 μM 5-AzadC and a bar graph showing relative SOST protein expression normalized to β-actin in 5-AzadC-treated normal chondrocytes (n = 3) (error bars = standard error, *p = 0.009). c DNA methylation and unmethylation status of the SOST promoter in cultured normal chondrocytes after treatment with 5 μM 5-AzadC. d DNA methylation values in CpG-rich region of the SOST promoter in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC by quantitative methylation-specific PCR (error bars = standard error, *p = 0.032)
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Fig3: Effect of 5-AzadC treatment on SOST expression and DNA methylation status in the CpG-rich region of the SOST promoter. a Quantitative SOST mRNA expression in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.041). b Representative western blot of SOST protein levels in cultured normal chondrocytes after treatment with 5 μM 5-AzadC and a bar graph showing relative SOST protein expression normalized to β-actin in 5-AzadC-treated normal chondrocytes (n = 3) (error bars = standard error, *p = 0.009). c DNA methylation and unmethylation status of the SOST promoter in cultured normal chondrocytes after treatment with 5 μM 5-AzadC. d DNA methylation values in CpG-rich region of the SOST promoter in cultured normal chondrocytes (n = 3) after treatment with 5 μM 5-AzadC by quantitative methylation-specific PCR (error bars = standard error, *p = 0.032)
Mentions: To confirm the causal inverse association between SOST promoter methylation and gene expression, we evaluated the ability of the demethylating agent 5-AzadC to promote SOST expression in chondrocytes. Normal chondrocytes, for which the promoter was found to be methylated, were treated with 5-AzadC and subsequently we evaluated SOST mRNA and protein levels by real-time PCR and western blot analysis, respectively. We found that SOST mRNA and protein levels (p = 0.041, p = 0.009, respectively) were markedly increased in 5-AzadC-treated cells compared to untreated (Fig. 3a, b) and this 5-AzadC-induced change in gene expression was associated with a decrease in DNA methylation in the CpG-rich region of the SOST promoter (p = 0.032) (Fig. 3c, d).Fig. 3

Bottom Line: Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known.We observed that SOST's expression was upregulated in OA chondrocytes compared to normal.Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytogenetics and Molecular Genetics, University of Thessaly, Faculty of Medicine, Biopolis, Larissa, 41500, Greece. ioanna_papathanasiou@yahoo.gr.

ABSTRACT

Introduction: Sclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes' hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST's expression in OA chondrocytes.

Methods: SOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5'-Aza-2-deoxycytidine (5-AzadC) on SOST's expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).

Results: We observed that SOST's expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.

Conclusions: We report novel findings that DNA methylation regulates SOST's expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes' expression observed in OA.

No MeSH data available.


Related in: MedlinePlus