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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.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).

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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Related In: Results  -  Collection

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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.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).

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