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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

DNA methylation status of the SOST promoter in normal and osteoarthritis (OA) chondrocytes. a DNA methylation status of CpG island located upstream of the transcript start site (TSS) in the region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 14) (error bars = standard error, *p = 0.05 versus normal unmethylation status, **p = 0.043 versus normal methylation status). b DNA methylation values in the CpG-rich region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 10) by qMSP (error bars = standard error, *p = 0.004). c Schematic representation of the CpG-rich region of the SOST promoter showing the six CpG sites that were analyzed using the bisulfite DNA sequencing method. d The percentage of cytosine methylation of each CpG site located at the CpG region of the SOST promoter in cultured normal (n = 5) and OA chondrocytes (n = 7), after bisulfite DNA sequencing analysis (error bars = standard error, *p = 0.05 and **p = 0.046)
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Fig2: DNA methylation status of the SOST promoter in normal and osteoarthritis (OA) chondrocytes. a DNA methylation status of CpG island located upstream of the transcript start site (TSS) in the region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 14) (error bars = standard error, *p = 0.05 versus normal unmethylation status, **p = 0.043 versus normal methylation status). b DNA methylation values in the CpG-rich region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 10) by qMSP (error bars = standard error, *p = 0.004). c Schematic representation of the CpG-rich region of the SOST promoter showing the six CpG sites that were analyzed using the bisulfite DNA sequencing method. d The percentage of cytosine methylation of each CpG site located at the CpG region of the SOST promoter in cultured normal (n = 5) and OA chondrocytes (n = 7), after bisulfite DNA sequencing analysis (error bars = standard error, *p = 0.05 and **p = 0.046)

Mentions: Taking into consideration recent reports that demonstrated involvement of epigenetics in the regulation of SOSTs expression, we tested whether the DNA methylation status of the SOST promoter is different between normal and OA chondrocytes. Bioinformatic analysis revealed the presence of two CpG islands surrounding the TSS of the SOST gene. One CpG island was located upstream of the TSS and the other within exon 1, downstream of the transcription start codon. We then tested the CpG islands for the presence of transcription factors binding sites and especially Smad binding sites, as we wanted to investigate the role of BMP-2, mediated through Smad proteins, on SOST expression. As we found Smad binding sites only in the first CpG island located upstream of the TSS in the region of the SOST promoter, we investigated the methylation status of this CpG island. This CpG island was identified between −516 bp and −256 bp upstream of the TSS, as a region of DNA spanning over 200 bp with a GC content over 50 %. Using MSP technology, we evaluated the methylation status of this region in genomic DNA isolated from normal and OA chondrocytes. Our results showed a significant difference in the methylation status between normal and OA chondrocytes in this CpG island located at the SOST promoter (Fig. 2a). Moreover, analysis by qMSP demonstrated that this CpG-rich region at the SOST promoter was highly methylated in normal chondrocytes (76,18 % ± 0,842) compared to OA chondrocytes (72,68 % ± 0,654) (p = 0.004) (Fig. 2b). To identify the particular CpG sites in this region whose methylation status is associated with SOST expression, we analyzed six CpG dinucleotides in the CpG island located at the SOST promoter using bisulfite DNA sequencing. We found that CpG sites 1, 4 and 6 were highly methylated (p = 0.05, p = 0.05 and p = 0.046, respectively) in normal compared to OA chondrocytes (Fig. 2c and d).Fig. 2


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)

DNA methylation status of the SOST promoter in normal and osteoarthritis (OA) chondrocytes. a DNA methylation status of CpG island located upstream of the transcript start site (TSS) in the region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 14) (error bars = standard error, *p = 0.05 versus normal unmethylation status, **p = 0.043 versus normal methylation status). b DNA methylation values in the CpG-rich region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 10) by qMSP (error bars = standard error, *p = 0.004). c Schematic representation of the CpG-rich region of the SOST promoter showing the six CpG sites that were analyzed using the bisulfite DNA sequencing method. d The percentage of cytosine methylation of each CpG site located at the CpG region of the SOST promoter in cultured normal (n = 5) and OA chondrocytes (n = 7), after bisulfite DNA sequencing analysis (error bars = standard error, *p = 0.05 and **p = 0.046)
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Fig2: DNA methylation status of the SOST promoter in normal and osteoarthritis (OA) chondrocytes. a DNA methylation status of CpG island located upstream of the transcript start site (TSS) in the region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 14) (error bars = standard error, *p = 0.05 versus normal unmethylation status, **p = 0.043 versus normal methylation status). b DNA methylation values in the CpG-rich region of the SOST promoter in cultured normal (n = 10) and OA chondrocytes (n = 10) by qMSP (error bars = standard error, *p = 0.004). c Schematic representation of the CpG-rich region of the SOST promoter showing the six CpG sites that were analyzed using the bisulfite DNA sequencing method. d The percentage of cytosine methylation of each CpG site located at the CpG region of the SOST promoter in cultured normal (n = 5) and OA chondrocytes (n = 7), after bisulfite DNA sequencing analysis (error bars = standard error, *p = 0.05 and **p = 0.046)
Mentions: Taking into consideration recent reports that demonstrated involvement of epigenetics in the regulation of SOSTs expression, we tested whether the DNA methylation status of the SOST promoter is different between normal and OA chondrocytes. Bioinformatic analysis revealed the presence of two CpG islands surrounding the TSS of the SOST gene. One CpG island was located upstream of the TSS and the other within exon 1, downstream of the transcription start codon. We then tested the CpG islands for the presence of transcription factors binding sites and especially Smad binding sites, as we wanted to investigate the role of BMP-2, mediated through Smad proteins, on SOST expression. As we found Smad binding sites only in the first CpG island located upstream of the TSS in the region of the SOST promoter, we investigated the methylation status of this CpG island. This CpG island was identified between −516 bp and −256 bp upstream of the TSS, as a region of DNA spanning over 200 bp with a GC content over 50 %. Using MSP technology, we evaluated the methylation status of this region in genomic DNA isolated from normal and OA chondrocytes. Our results showed a significant difference in the methylation status between normal and OA chondrocytes in this CpG island located at the SOST promoter (Fig. 2a). Moreover, analysis by qMSP demonstrated that this CpG-rich region at the SOST promoter was highly methylated in normal chondrocytes (76,18 % ± 0,842) compared to OA chondrocytes (72,68 % ± 0,654) (p = 0.004) (Fig. 2b). To identify the particular CpG sites in this region whose methylation status is associated with SOST expression, we analyzed six CpG dinucleotides in the CpG island located at the SOST promoter using bisulfite DNA sequencing. We found that CpG sites 1, 4 and 6 were highly methylated (p = 0.05, p = 0.05 and p = 0.046, respectively) in normal compared to OA chondrocytes (Fig. 2c and d).Fig. 2

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