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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

SOST mRNA and protein expression levels in normal and osteoarthritis (OA) chondrocytes. a Quantitative SOST mRNA expression in cultured normal (n = 10) and OA chondrocytes (n = 14). GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.005). b Representative western blot of SOST protein expression in cultured normal and OA chondrocytes and a bar graph showing relative SOST protein expression normalized to β-actin in normal (n = 5) and OA chondrocytes (n = 5) (error bars = standard error, *p = 0.038)
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Fig1: SOST mRNA and protein expression levels in normal and osteoarthritis (OA) chondrocytes. a Quantitative SOST mRNA expression in cultured normal (n = 10) and OA chondrocytes (n = 14). GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.005). b Representative western blot of SOST protein expression in cultured normal and OA chondrocytes and a bar graph showing relative SOST protein expression normalized to β-actin in normal (n = 5) and OA chondrocytes (n = 5) (error bars = standard error, *p = 0.038)

Mentions: Using real-time PCR, we found higher SOST mRNA expression levels in OA compared to normal chondrocytes (p = 0.005) (Fig. 1a). SOST protein levels confirmed our real-time PCR findings. Western blot analysis revealed that SOST protein expression is elevated in OA chondrocytes compared to normal (p = 0.038) (Fig. 1b).Fig. 1


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)

SOST mRNA and protein expression levels in normal and osteoarthritis (OA) chondrocytes. a Quantitative SOST mRNA expression in cultured normal (n = 10) and OA chondrocytes (n = 14). GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.005). b Representative western blot of SOST protein expression in cultured normal and OA chondrocytes and a bar graph showing relative SOST protein expression normalized to β-actin in normal (n = 5) and OA chondrocytes (n = 5) (error bars = standard error, *p = 0.038)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491261&req=5

Fig1: SOST mRNA and protein expression levels in normal and osteoarthritis (OA) chondrocytes. a Quantitative SOST mRNA expression in cultured normal (n = 10) and OA chondrocytes (n = 14). GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.005). b Representative western blot of SOST protein expression in cultured normal and OA chondrocytes and a bar graph showing relative SOST protein expression normalized to β-actin in normal (n = 5) and OA chondrocytes (n = 5) (error bars = standard error, *p = 0.038)
Mentions: Using real-time PCR, we found higher SOST mRNA expression levels in OA compared to normal chondrocytes (p = 0.005) (Fig. 1a). SOST protein levels confirmed our real-time PCR findings. Western blot analysis revealed that SOST protein expression is elevated in OA chondrocytes compared to normal (p = 0.038) (Fig. 1b).Fig. 1

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