Zoledrinic Acid Induces Steoblastic Differentiation of Mesenchymal Stem Cells without Change in Hypomethylation Status of OSTERIX Promoter.

OBJECTIVE

Mechanism of zoledronic acid on osteoblastic differentiation of mesenchymal stem cells (MSCs) has not fully understood. With the knowledge of some drugs mechanism that alter methylation pattern of some genes, the present research sets out to evaluate osterix (OSX) promoter methylation pattern during zoledronic acid-induced osteoblastic differentiation of MSCs.

MATERIALS AND METHODS

In this experimental study, MSCs were isolated from human bone marrow. For osteogenic differentiation, MSCs were pulse treated with 5ìM Zoledronic acid for 3 hours and incubated after a medium change in osteogenic differentiation medium for 3 weeks. DNA and RNA were extracted on days 0, 7, 14 and 21 of MSCs differentiating to osteoblast. After cDNA synthesis, OSX expression was evaluated by RT-PCR and quantitative Real-Time PCR. After multiplicity of infection (MOI) treatment, gene specific methylation of OSX was analyzed by methylation specific PCR (MSP).

RESULTS

The mRNA expression of OSX was increased in osteoblast differentiated cells induced by zoledronic acid, especially on days 14 and 21 of differentiation (p<0.05), but expression of OSX didn't change in undifferentiated MSCs. MSP revealed that, on day 0, undifferentiated MSCs are totally methylated. But, on day 7 of differentiation, MSCs treated by zoledronic acid were totally unmethylated. OSX promoter remained unmethylated, afterwards.

CONCLUSION

MSP revealed that OSX had a dynamic pattern in methylation, while MSCs gradually differentiated to osteoblasts. Our finding showed that promoter region of OSX is hypomethylated independently from zoledronic acid treatment during osteoblastic differentiation. This knowledge is important to understand drug mechanisms and can be useful for developing new therapies to combat against bone diseases.