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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 bone morphogenic protein 2 (BMP-2) and 5-AzadC treatment on SOST expression. a and b Efects of BMP-2 on SOST mRNA expression levels in cultured normal (n = 3) and osteoarthritis (OA) chondrocytes (n = 3). Osteocalcin and LRP-5 used as positive controls that upregulated by BMP-2 and LRP-6 and MMP-7 used as negative controls that not regulated by BMP-2(error bars = standard error, *p = 0.004). c Detection of SOST mRNA expression levels by real time PCR after BMP-2 treatment in cultured normal chondrocytes (n = 3) with or without 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.001 versus control and DMSO/BMP-2 treatment). MMP matrix metalloproteinase, LRP low-density lipoprotein receptor-related protein
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Fig4: Effect of bone morphogenic protein 2 (BMP-2) and 5-AzadC treatment on SOST expression. a and b Efects of BMP-2 on SOST mRNA expression levels in cultured normal (n = 3) and osteoarthritis (OA) chondrocytes (n = 3). Osteocalcin and LRP-5 used as positive controls that upregulated by BMP-2 and LRP-6 and MMP-7 used as negative controls that not regulated by BMP-2(error bars = standard error, *p = 0.004). c Detection of SOST mRNA expression levels by real time PCR after BMP-2 treatment in cultured normal chondrocytes (n = 3) with or without 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.001 versus control and DMSO/BMP-2 treatment). MMP matrix metalloproteinase, LRP low-density lipoprotein receptor-related protein

Mentions: BMP-2 has been reported to play a significant role in the regulation of SOST expression in bone. To gain insight into the molecular mechanisms underlying SOST expression in OA chondrocytes, we examined the effect of BMP-2 on SOST expression in normal and OA chondrocytes. We found that BMP-2 treatment resulted in significant induction of SOST expression in OA chondrocytes (p = 0.004), but not in normal chondrocytes (Fig. 4a and b). However, SOST expression levels were upregulated after BMP-2 treatment in 5-AzadC-treated normal chondrocytes (Fig. 4c) (p = 0.001). To further investigate the intracellular signaling pathway involved in BMP-2-induced SOST expression, normal, OA, BMP-2 and/or 5-AzadC-treated chondrocytes were subjected to ChIP assay using an antibody against Smad-1/5/8 and we tested whether Smads bind to the SOST promoter via Smad binding elements. We found that the SOST promoter contains a conserved Smad binding site in the CpG island located upstream of the TSS and that Smad1/5/8 binding was enhanced in OA compared to normal chondrocytes (p = 0.05) and in BMP-2-treated OA compared to untreated chondrocytes (p = 0.05) (Fig. 5a and c). Moreover, stronger binding of Smad1/5/8 was observed in 5-AzadC-treated normal chondrocytes compared to untreated (Fig. 5b) (p = 0.05) and this affinity was significantly increased in BMP-2/5-AzadC-treated normal chondrocytes compared to 5-AzadC-treated, BMP-2-treated and untreated chondrocytes (Fig. 5d and e) (p = 0.05). No difference was observed between BMP-2-treated and untreated normal chondrocytes (Fig. 5c).Fig. 4


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 bone morphogenic protein 2 (BMP-2) and 5-AzadC treatment on SOST expression. a and b Efects of BMP-2 on SOST mRNA expression levels in cultured normal (n = 3) and osteoarthritis (OA) chondrocytes (n = 3). Osteocalcin and LRP-5 used as positive controls that upregulated by BMP-2 and LRP-6 and MMP-7 used as negative controls that not regulated by BMP-2(error bars = standard error, *p = 0.004). c Detection of SOST mRNA expression levels by real time PCR after BMP-2 treatment in cultured normal chondrocytes (n = 3) with or without 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.001 versus control and DMSO/BMP-2 treatment). MMP matrix metalloproteinase, LRP low-density lipoprotein receptor-related protein
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: Effect of bone morphogenic protein 2 (BMP-2) and 5-AzadC treatment on SOST expression. a and b Efects of BMP-2 on SOST mRNA expression levels in cultured normal (n = 3) and osteoarthritis (OA) chondrocytes (n = 3). Osteocalcin and LRP-5 used as positive controls that upregulated by BMP-2 and LRP-6 and MMP-7 used as negative controls that not regulated by BMP-2(error bars = standard error, *p = 0.004). c Detection of SOST mRNA expression levels by real time PCR after BMP-2 treatment in cultured normal chondrocytes (n = 3) with or without 5-AzadC. GAPDH was used for normalization of the real-time PCR data (error bars = standard error, *p = 0.001 versus control and DMSO/BMP-2 treatment). MMP matrix metalloproteinase, LRP low-density lipoprotein receptor-related protein
Mentions: BMP-2 has been reported to play a significant role in the regulation of SOST expression in bone. To gain insight into the molecular mechanisms underlying SOST expression in OA chondrocytes, we examined the effect of BMP-2 on SOST expression in normal and OA chondrocytes. We found that BMP-2 treatment resulted in significant induction of SOST expression in OA chondrocytes (p = 0.004), but not in normal chondrocytes (Fig. 4a and b). However, SOST expression levels were upregulated after BMP-2 treatment in 5-AzadC-treated normal chondrocytes (Fig. 4c) (p = 0.001). To further investigate the intracellular signaling pathway involved in BMP-2-induced SOST expression, normal, OA, BMP-2 and/or 5-AzadC-treated chondrocytes were subjected to ChIP assay using an antibody against Smad-1/5/8 and we tested whether Smads bind to the SOST promoter via Smad binding elements. We found that the SOST promoter contains a conserved Smad binding site in the CpG island located upstream of the TSS and that Smad1/5/8 binding was enhanced in OA compared to normal chondrocytes (p = 0.05) and in BMP-2-treated OA compared to untreated chondrocytes (p = 0.05) (Fig. 5a and c). Moreover, stronger binding of Smad1/5/8 was observed in 5-AzadC-treated normal chondrocytes compared to untreated (Fig. 5b) (p = 0.05) and this affinity was significantly increased in BMP-2/5-AzadC-treated normal chondrocytes compared to 5-AzadC-treated, BMP-2-treated and untreated chondrocytes (Fig. 5d and e) (p = 0.05). No difference was observed between BMP-2-treated and untreated normal chondrocytes (Fig. 5c).Fig. 4

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